Toxin-related antibodies with antimicrobial and antiviral activity

ABSTRACT

Anti-idiotypic antibodies which recognise the idiotope of an antibody specific for a yeast killer toxin possess microbicidal activity. Fragments (e.g. decapeptides) of these anti-idiotypic antibodies, particularly those comprising CDR residues, also show microbicidal activity, as do peptides having 5 the same sequence but composed of D-amino acids, or including amino acid substitutions. Peptidomimetics of these microbicidal polypeptides are also provided. Antiviral activity is also seen.

TECHNICAL FIELD

This invention is in the field of microbicides and antivirals, inparticular those derived from yeast killer toxins.

BACKGROUND ART

Killer toxins (KTs) are proteins secreted by yeasts which are able tokill other yeasts or microorganisms which compete in nature for the sameecological niche [1]. Although they are attractive therapeutic tools,due to their wide spectrum of microbicidal activity, they are of nopractical use because of their instability in the host physiologicalmilieu as well as their antigenicity and toxicity. Instead, the use ofanti-idiotypic antibodies which mimic KTs has been shown to beeffective.

The killer toxin from Pichia anomala (‘PaKT’ ) has a wide spectrum ofmicrobicidal activity against pathogens including Candida albicans,Aspergillus fumigatus, Pneumocystis carinii, Mycobacterium tuberculosis,Pseudomonas aeruginosa, and Staphylococcus aureus [2, 3, 4]. Thisobservation has been exploited by the generation of a PaKT-neutralizingmonoclonal antibody in mice (mAb KT4) [5] whose idiotype (Id) is able toinduce the production of anti-idiotypic antibodies (antilds) [6, 7, 8].These antilds represent the internal image of the active PaKT domain andas such exert its biological activities, including binding to the PaKTreceptor (KTR) of susceptible microorganisms and broad spectrummicrobicidal activity overlapping that of PaKT (FIG. 1).

Experimental animals in which these antibodies (‘KTIdAb ) are raised byidiotypic vaccination with mAb KT4 have repeatedly been shown to beprotected against mucosal or systemic challenges by C.albicans [7, 8].There is also ample evidence of susceptibility in vitro to KTIdAb bydiverse microbial pathogens such as M.tuberculosis (including multidrugresistant strains), P.carinii, and others [4, 9].

Idiotypic theory (FIG. 1) also predicted that antibodies against PaKTreceptors would mimic PaKT activity. This has been demonstrated inanimals and humans during the course of experimental and naturalinfections caused by PaKT-sensitive microorganisms [10]. Human naturalanti-KTR antibodies have been shown to have microbicidal activity invitro against C.albicans, M.tuberuclosis, and P.carinii, to inhibitP.carinii infectivity of nude rats, and to be protective against passivetransfer in vivo, in an experimental model of rat vaginal candidiasis[4, 10, 11].

Based on these results, and in order to obtain standard KTldAb insufficient amounts, rat monoclonal IgM (mAb K10) and mouse single-chainFv (scFv H6) microbicidal antibodies have been obtained [12, 13]. Thesetwo antibodies have strong microbicidal effects in vitro againstimportant pathogenic microorganisms including: C.albicans [12, 13];C.krusei and C.glabrata (including fluconazole-resistant strains);Cryptococcus neoformans; A. fumigalus [14]; M.tuberculosis [4];S.aureus, Enterococcus faecalis, E.faecium, and Streptococcus pneumoniae(including methicillin-, vancomycin- and penicillin-resistant strains)[15], S.mutans, Leishmania major, L.infantum and Achantamoebacastellani. Furthermore, they showed specific therapeutic activity in anexperimental model of rat vaginal candidiasis by intravaginaladministration [13]. In addition, K10 proved to be therapeutic againstP.carinii pneumonia in rats infected by aerosol administration [16], andin mice transplanted with T cell depleted bone marrow againstaspergillosis caused by nasal instillation [14].

Although the existence of scFv H6 has been reported, a method for itsmanufacture has not previously been disclosed, and nor has its aminoacid sequence.

It is an object of the invention to provide further and improvedantimicrobial and/or antiviral compounds.

DISCLOSURE OF THE INVENTION

Antibodies of the Invention

The invention provides an anti-idiotypic antibody which recognises theidiotope of an antibody specific for a yeast killer toxin, with theproviso that the anti-idiotypic antibody is not the K10 rat monoclonalantibody. The antibody preferably has microbicidal activity (e.g. itretains yeast killer toxin activity) and/or antiviral activity.

An anti-idiotypic antibody of the invention can be used to generatefurther anti-idiotypic antibodies (anti-anti-anti-idiotypic with respectto the original killer toxin). The anti-anti-anti-idiotypic antibody ofthe invention can in turn be used to generate further anti-idiotypicantibodies (anti-anti-anti-anti-anti-idiotypic with respect to theoriginal killer toxin). Thus the microbicidal activity of the killertoxin can be transferred through successive generations ofanti-idiotypic antibodies, and these various generations are within thescope of the invention.

Thus the invention provides an antibody which recognises the idiotope ofan anti-idiotypic antibody of the invention (i.e. it provides ananti-anti-anti-idiotypic antibody), an antibody which recognises theidiotope of such an anti-anti-anti-idiotypic antibody (i.e. ananti-anti-anti-anti-anti-idiotypic antibody) etc. In general, therefore,the invention provides an antibody which recognises the idiotope of an(anti-)_(n)-idiotypic antibody of the invention, wherein n is an oddnumber (e.g. 1, 3, 5, 7, 9 etc.). These antibodies will generally bindto the idiotope of an anti-toxin antibody such as KT4 and preferablyhave microbicidal and/or antiviral activity.

For production of these (anti-)_(n)-idiotypic antibodies, the inventionprovides (anti-)_(m)-idiotypic antibodies , wherein m is an even number(e.g. 2, 4, 6, 8 etc.). These antibodies will generally bind to a killertoxin.

The term ‘antibody’ includes any of the various natural and artificialantibodies and antibody-derived proteins which are available. Thus theterm ‘antibody’ includes polyclonal antibodies, monoclonal antibodies,Fab fragments, F(ab′)₂ fragments, Fv fragments, single-chain Fv (scFV)antibodies, oligobodies, etc.

To increase compatibility with the human immune system, it is preferredto use human antibodies. As an alternative, antibodies of the inventionmay be chimeric or humanized versions of non-human antibodies [e.g.refs. 17 & 18 ].

In chimeric antibodies, non-human constant regions are substituted byhuman constant regions but variable regions remain non-human.

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting complementarity determining regions (CDRs)from the non-human variable region onto a human framework(“CDR-grafting”), with the optional additional transfer of one or moreframework residues from the non-human antibody (“humanizing”); (2)transplanting entire non-human variable domains, but “cloaking” themwith a human-like surface by replacement of surface residues (“veneering”). In the present invention, humanized antibodies include thoseobtained by CDR-grafting, humanizing, and veneering or variable regions.[e.g. refs. 19 to 25].

The constant regions of humanized antibodies are derived from humanimmunoglobulins. The heavy chain constant region can be selected fromany of the five isotypes: α, δ, ε, γ or μ.

Humanized or fully-human antibodies can also be produced usingtransgenic animals that are engineered to contain human immunoglobulinloci. For example, ref. 26 discloses transgenic animals having a humanlg locus wherein the animals do not produce functional endogenousimmunoglobulins due to the inactivation of endogenous heavy and lightchain loci. Ref. 27 also discloses transgenic non-primate mammalianhosts capable of mounting an immune response to an immunogen, whereinthe antibodies have primate constant and/or variable regions, andwherein the endogenous immunoglobulin-encoding loci are substituted orinactivated. Ref. 28 discloses the use of the Cre/Lox system to modifythe immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. Ref. 29 discloses non-human mammalian hosts having inactivatedendogenous lg loci and functional human Ig loci. Ref. 30 disclosesmethods of making transgenic mice in which the mice lack endogenousheavy chains, and express an exogenous immunoglobulin locus comprisingone or more xenogeneic constant regions.

Antibodies of the invention may include a label. The label may bedetectable directly, such as a radioactive or fluorescent label.Alternatively, the label may be detectable indirectly, such as an enzymewhose products are detectable (e.g. luciferase, β-galactosidase,peroxidase etc.) or a binding partner such as biotin and avidin orstreptavidin.

Antibodies of the invention may be attached to a solid support.

Antibodies of the invention may be produced by any suitable means (e.g.by recombinant expression).

Preferred antibodies of the invention are single chain Fv antibodies.These may be produced by joining heavy and light chain variable regionsfrom a starting monoclonal antibody of interest, or may be identified byscreening a scFv library (e.g. by phage display). Reference 31 disclosesa phage display method for producing anti-idiotypic scFv antibodies.

Particularly preferred scFv antibodies of the invention are H6 (SEQ IDs1 & 2) and H20 (SEQ IDs 21 & 22). Antibodies comprising one or more(e.g. 2, 3, 4, 5 or 6) of the CDRs from H6 and H20 are also preferred,as are derivatives of H6 and H20 in which: (a) one or more (e.g. 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) framework residues aresubstituted with other amino acids; (b) the linker sequence (SEQ ID 30)is replaced with an alternative linker sequence; (c) the ‘E-tag’sequence (SEQ ID 59) is omitted or replaced. Fusion proteins comprisingH6 or H20, or derivatives (a) to (c), at the N- or C-terminus are alsouseful. The H6 and H20 CDRs may optionally contain 1, 2, 3 or 4 aminoacid substitutions.

Other preferred antibodies of the invention are humanized antibodies.Particularly preferred humanized antibodies of the invention compriseone or more (e.g. 2, 3, 4, 5 or 6) CDRs from H6, H20 or K20.

Polypeptides and Antibodyfragments

It has surprisingly been found that short fragments (e.g. 10merfragments) of the variable regions of anti-idiotypic antibodies of theinvention can retain the antibodies' KT-like microbicidal activity. Evenmore surprisingly, L-amino acids in these fragments can be replaced withD-amino acids without removing microbicidal activity, and amino acidswithin the fragments can be substituted with other amino acids withoutremoving microbicidal activity. In addition, the fragments have beenfound to possess anti-viral activity.

Thus the invention provides a polypeptide comprising: at least one aminoacid sequence which is a fragment of at least x amino acids from theamino acid sequence of a variable region of an antibody of theinvention, optionally with y amino acid(s) within said x amino acidsbeing substituted by different amino acid(s). The polypeptide preferablyhas microbicidal and/or antiviral activity.

The polypeptide preferably consists of no more than 250 amino acids(e.g. no more than 225, 200, 190, 180, 170, 160, 150, 140, 130, 120,110, 100, 95, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or even 5 amino acids).Polypeptides consisting of between 5 and 90 amino acids are preferred(e.g. consisting of between 5 and 80, 5 and 70, 5 and 60 amino acidsetc.). Particularly preferred are polypeptides consisting of between 8and 25 amino acids are preferred.

The polypeptide preferably consists of at least 3 amino acids (e.g. atleast 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, or at least200 amino acids).

The value of x is preferably at least 3 (e.g. at least 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,60, 70, 80, 90, 100, 125, 150, 175, or at least 200).

The value of y will be less than x and, depending on the value of x, itmay be x-1, x-2, x-3, x-4, x-5, x-6, x-7, x-8, x-9, x-10, x- 11, x-12,x-13, x-14, or x-15. Preferred values of y are 1, 2, 3, 4 and 5. The yamino acids are typically substituted by A, C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W, or Y. Each of the y substitutions may be thesame or different as the others. The substitution is preferably by G or,more preferably, by A [32, 33]. The substituting amino acid may be an L-or a D- amino acid but, where the other x amino acids all share a singlestereo-configuration (i.e. all D- or all L-), it preferably has thatstereo-configuration (although, of course, G has no stereoisomers).

Where the fragment of x amino acids includes a C, the value of y ispreferably at least 1 such that the C is substituted for another aminoacid, such as S. Removal of C in this way improves resistance tooxidation.

The fragment of at least x amino acids preferably includes at least zamino acids from a CDR within the antibody. The value of z is preferablyat least 1 (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more).

The polypeptide may comprise more than one (e.g. 2, 3, 4, 5, 6) aminoacid sequence each of which is a fragment of at least x amino acids(e.g. SEQ ID 25). In such polypeptides, the value of x in each fragmentmay be the same or different, the value of y within each x may be thesame or different, and the value of z within each x may be the same ordifferent. The fragments may be joined by linker peptides such asglycine-rich linker sequences (e.g. SEQ ID 30).

The invention also provides a polypeptide having formula NH₂—A—B—C—COOH,wherein: A is a polypeptide sequence consisting of a amino acids; C is apolypeptide sequence consisting of c amino acids; B is a polypeptidesequence which is, as defined above, a fragment of at least x aminoacids from the amino acid sequence of a variable region of an antibodyof the invention, optionally with y amino acids within said x aminoacids being substituted by different amino acid(s). The polypeptidepreferably has microbicidal and/or antiviral activity.

The value of a is generally at least 1 (e.g. at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250,300, 350, 400, 450, 500 etc.). The value of c is generally at least 1(e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13,14,15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 etc.). The value ofa+c is at least 1 (e.g. at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500etc.). It is preferred that the value of a+c is at most 1000 (e.g. atmost 900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 190, 180,170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2).

The amino acid sequence of —A— typically shares less than m% sequenceidentity to the a amino acids which are N-terminal of sequence —B— in avariable region of an antibody of the invention (e.g. in SEQ ID 2), andthe amino acid sequence of —C— typically shares less than n% sequenceidentity to the c amino acids which are C-terminal of sequence —B— in avariable region of an antibody of the invention (e.g. in SEQ ID 2). Ingeneral, the values of m and n are both 60 or less (e.g. 50, 40, 30, 20,10 or less). The values of m and n may be the same as or different fromeach other.

The polypeptide may comprise a mimotope of a yeast killer toxin.

Preferred polypeptides comprise sequenceAA₁-AA₂AA₃-AA₄-AA₅-AA₆-AA₇-AA₈-AA₉-AA₁₀, wherein: each of AA₁ . . . AA₁₀is independently a D- or L- amino acid; AA₁ is E, A or G; AA₂ is K, A orG; AA₃ is V, A or G; AA₄ is T, A or G; AA₅ is M, A or G; AA₇ is T, A orG; AA₇ is C, S, A or G; AA₈ is S, A or G; AA₉ is A or G; and AA₁₀ is S,A or G; provided that no more than p of AA₁, AA_(2,) AA₃, AA_(4,)AA_(5,) AA_(6,) AA_(7,) AA_(8,) AA₉, and AA₁₀ are A; and provided thatno more than q of AA₁, AA_(2,) AA_(3,) AA_(4,) AA_(5,) AA_(6,) AA_(7,)AA₈, AA₉, and AA₁₀ are G. The value of p is 1, 2, 3 or 4, and ispreferably 1 or 2. The value of q is 0, 1 or 2 and is preferably 0 (i.e.no glycine residues) or 1.

Particularly preferred polypeptides comprise or consist of amino acidsequences SEQ IDs : 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18,19, 20, 23, 24, 25, 26, 27, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70 & 71, with constituent amino acids inthe D- and/or L- configuration. SEQ IDs 3, 4, 23, 27 and 33 are mostpreferred.

For reference, the values of x, y and z for various of these polypeptidesequences are as follows: SEQ ID x y z 3 10 0 3 4 10 1 3 5 10 1 3 6 10 13 7 10 1 3 8 10 1 3 9 10 1 3 10 10 1 3 11 10 1 3 12 10 1 3 14 9 1 2 15 81 1 16 7 1 0 17 6 1 0 18 5 1 0 19 4 1 0 20 3 1 0 23 10 2 3 24 10 0 3 2615 0 8 27 9 0 8 32 10 2 3 33 10 2 3Further microbicidal and/or antiviral fragments can be identified byscreening panels of overlapping peptide fragments once the sequence ofan anti-idiotypic antibody (e.g. H6, H20, K20) is known e.g. by usingthe PepScan method [34]. Overlapping fragments of 4mers, 5mers, 6mers,7mers, 8mers, 9mers, 10 mers, 11 mers, 12mers, 13mers etc. can be testedfor microbicidal ability without difficulty e.g. using the in vitroassays disclosed in the examples herein.

Polypeptides of the invention may be linear, branched or cyclic [35] butthey are preferably linear chains of amino acids. Where cysteineresidues are present, polypeptides of the invention may be linked toother polypeptides via disulfide bridges (and, in particular, linked toother polypeptides of the invention to form homodimers or heterodimers).Polypeptides of the invention may comprise L-amino acids and/or D-aminoacids. The inclusion of D-amino acids is preferred in order to conferresistance to mammalian proteases.

Polypeptide Production

Polypeptides of the invention may be produced by various means.

A preferred method for production involves in vitro chemical synthesis[36, 37]. Solid-phase peptide synthesis is particularly preferred, suchas methods based on t-Boc or Fmoc [38] chemistry. Enzymatic synthesis[39] may also be used in part or in full.

As an alternative to chemical synthesis, biological synthesis may beused e.g. the polypeptides may be produced by translation. This may becarried out in vitro or in vivo. Biological methods are in generalrestricted to the production of polypeptides based on L-amino acids, butmanipulation of translation machinery (e.g. of aminoacyl-tRNA molecules)can be used to allow the introduction of D-amino acids (or of othernon-natural amino acids, such as iodotyrosine or methylphenylalanine,azidohomoalanine, etc.) [40]. Where D-amino acids are included in thepolypeptides of the invention, however, it is preferred to use chemicalsynthesis. Further details on polypeptide expression are given below.

To facilitate biological peptide synthesis, the invention providesnucleic acid that encodes a polypeptide of the invention. The inventionalso provides nucleic acid that encodes an antibody of the invention.

The nucleic acid may be DNA or RNA (or hybrids thereof), or theiranalogues, such as those containing modified backbones (e.g.phosphorothioates) or peptide nucleic acids (PNA). It may besingle-stranded (e.g. mRNA) or double-stranded, and the inventionincludes both individual strands of a double-stranded nucleic acid (e.g.for antisense, priming or probing purposes). It may be linear orcircular. It may be labelled. It may be attached to a solid support.

Nucleic acid according to the invention can, of course, be prepared inmany ways e.g. by chemical synthesis (e.g. phosphoramidite synthesis ofDNA) in whole or in part, by nuclease digestion of longer molecules, byligation of shorter molecules, from genomic or cDNA libraries, by use ofpolymerases etc.

The invention provides vectors (e.g. plasmids) comprising nucleic acidof the invention (e.g. expression vectors and cloning vectors) and hostcells (prokaryotic or eukaryotic) transformed with such vectors.

These vectors can also be used for nucleic acid immunisation [e.g. refs.41, 42, 43, 44, 45 etc.]. Peptides can be expressed in vivo in this way,as can therapeutic antibodies. It is also possible to express idiotopes(e.g. the KT4 idiotope) to elicit anti-idiotypic antibodies of theinvention in a patient in vivo.

Host cells which contain nucleic acid of the invention and which expresspolypeptide or antibody of the invention may be used as deliveryvehicles e.g. commensal bacteria [46]. This is particularly useful fordelivery to mucosal surfaces.

The Yeast Killer Toxin

Killer toxins were originally identified in Saccharomyces cerevisiae[47] and have since been identified in other genera of yeasts includingPichia (such as P.anomala, P.kluyveri and P.farinosa), Hanseniaspora(such as H.uvarum), Williopsis (such as W.mrakii), Candida (such asC.maltosa), Debaryomyces (such as D.hansenii), Schwanniomyces (such asS.occidentalis), Cryptococcus (such as C.humicola), Torulopsis (such asT.glabrata), Ustilago (such as U.maydis), Zygosaccharomyces (such asZ.bailii) and Kluyveromyces (such as K.lactis and K.phaffii).

Any of these various toxins can be used with the present invention.Preferred toxins are (a) those with a broad range of microbicidalactivity and (b) those from Pichia. A particularly preferred toxin isthe killer toxin from Pichia anomala (‘PaKT’).

Microbicidal Activity

The polypeptide or antibody of the invention preferably has microbicidalactivity.

Preferably, it has anti-mycotic activity and/or anti-bacterial activity.Anti-bacterial activity may be against a Gram-negative or Gram-positivebacterium.

More preferably, it has activity against a microbe which has aglucan-based cell wall.

Microbes which are susceptible to polypeptides and antibodies of theinvention include bacteria, fungi and protozoa, and include, but are notlimited to: Candida species, such as C.albicans; Cryptococcus species,such as C.neoformans; Enterococcus species, such as E.faecalis;Streptococcus species, such as S.pneumoniae, S.mutans, S.agalactiae andS.pyogenes; Leishmania species, such as L.major and L.infantum;Acanthamoeba species, such as A.castellani; Aspergillus species, such asA.fumigatus; Pneumocystis species, such as P.carinii; Mycobacteriumspecies, such as M.tuberculosis; Pseudomonas species, such asP.aeruginosa; Staphylococcus species, such as S.aureus; Salmonellaspecies, such as S.typhimurium; and Escherichia species, such as E.coli.

Antiviral Activity

The polypeptide or antibody of the invention preferably has antiviralactivity.

Preferably, it has antiviral activity against a human virus, such as amyxovirus (e.g. an orthomyxovirus) or a retrovirus (e.g. a lentivirus).

Viruses which are susceptible to polypeptides and antibodies of theinvention include, but are not limited to: influenza virus (A or B),human immunodeficiency virus (HIV-1, HIV-2, HIV-O), respiratorysyncytial virus (RSV), yellow fever virus, etc.

Drug Design and Peptidomimetics

Polypeptides of the invention are useful microbicides and antivirals intheir own right. However, they may be refined to improve microbicidaland/or antiviral activity (either general or specific) or to improvepharmacologically important features such as bio-availability,toxicology, metabolism, pharmacokinetics etc. The polypeptides maytherefore be used as lead compounds for further research and refinement.

Polypeptides of the invention can be used for designing peptidomimeticmolecules [e.g. refs. 48 to 53] with microbicidal and/or antiviralactivity. These will typically be isosteric with respect to thepolypeptides of the invention but will lack one or more of their peptidebonds. For example, the peptide backbone may be replaced by anon-peptide backbone while retaining important amino acid side chains.

The peptidomimetic molecule may comprise sugar amino acids [54].Peptoids may be used.

To assist in the design of peptidomimetic molecules, a pharmacophore(i.e. a collection of chemical features and 3D constraints thatexpresses specific characteristics responsible for activity) can bedefined for the KM peptides. The pharmacophore preferably includessurface-accessible features, more preferably including hydrogen bonddonors and acceptors, charged/ionisable groups, and/or hydrophobicpatches. These may be weighted depending on their relative importance inconferring activity [55].

Pharmacophores can be determined using software such as CATALYST(including HypoGen or HipHop) [56], CERIUS², or constructed by hand froma known conformation of a polypeptide of the invention. Thepharmacophore can be used to screen structural libraries, using aprogram such as CATALYST. The CLIX program [57] can also be used, whichsearches for orientations of candidate molecules in structural databasesthat yield maximum spatial coincidence with chemical groups whichinteract with the receptor.

The binding surface or pharmacophore can be used to map favourableinteraction positions for functional groups (e.g. protons, hydroxylgroups, amine groups, hydrophobic groups) or small molecule fragments.Compounds can then be designed de novo in which the relevant functionalgroups are located in substantially the same spatial relationship as inpolypeptides of the invention.

Functional groups can be linked in a single compound using eitherbridging fragments with the correct size and geometry or frameworkswhich can support the functional groups at favourable orientations,thereby providing a peptidomimetic compound according to the invention.Whilst linking of functional groups in this way can be done manually,perhaps with the help of software such as QUANTA or SYBYL, automated orsemi-automated de novo design approaches are also available, such as:

-   -   MCSS/HOOK [58, 59, 56], which links multiple functional groups        with molecular templates taken from a database.    -   LUDI [60, 56], which computes the points of interaction that        would ideally be fulfilled by a ligand, places fragments in the        binding site based on their ability to interact with the        receptor, and then connects them to produce a ligand.    -   MCDLNG [61], which fills a receptor binding site with a        close-packed array of generic atoms and uses a Monte Carlo        procedure to randomly vary atom types, positions, bonding        arrangements and other properties.    -   GROW [62], which starts with an initial ‘seed’ fragment (placed        manually or automatically) and grows the ligand outwards.    -   SPROUT [63], suite which includes modules to: identify        favourable hydrogen bonding and hydrophobic regions within a        binding pocket (HIPPO module); select functional groups and        position them at target sites to form starting fragments for        structure generation (EleFAnT); generate skeletons that satisfy        the steric constraints of the binding pocket by growing spacer        fragments onto the start fragments and then connecting the        resulting part skeletons (SPIDeR); substitute hetero atoms into        the skeletons to generate molecules with the electrostatic        properties that are complementary to those of the receptor site        (MARABOU). The solutions can be clustered and scored using the        ALLigaTOR module.    -   CAVEAT [64], which designs linking units to constrain acyclic        molecules.    -   LEAPFROG [65], which evaluates ligands by making small stepwise        structural changes and rapidly evaluating the binding energy of        the new compound. Changes are kept or discarded based on the        altered binding energy, and structures evolve to increase the        interaction energy with the receptor.    -   GROUPBUILD [66], which uses a library of common organic        templates and a complete empirical force field description of        the non-bonding interactions between a ligand and receptor to        construct ligands that have chemically reasonable structure and        have steric and electrostatic properties complimentary to the        receptor binding site.    -   RASSE [67]

These methods identify microbicidal compounds. These compounds may bedesigned de novo, may be known compounds, or may be based on knowncompounds. The compounds may be useful microbicides and/or antiviralsthemselves, or they may be prototypes which can be used for furtherpharmaceutical refinement (i.e. lead compounds) in order to improvebinding affinity or other pharmacologically important features (e.g.bio-availability, toxicology, metabolism, pharmacokinetics etc.).

The invention thus provides: (i) a compound identified using these drugdesign methods; (ii) a compound identified using these drug designmethods, for use as a pharmaceutical; (iii) the use of a compoundidentified using these drug design methods in the manufacture of amicrobicide and/or an antiviral; and (iv) a method of treating a patientwith a microbial or viral infection, comprising administering aneffective amount of a compound identified using these drug designmethods.

As well as being useful compounds individually, ligands identified insilico by the structure-based design techniques can also be used tosuggest libraries of compounds for ‘traditional’ in vitro or in vivoscreening methods. Important pharmaceutical motifs in the ligands can beidentified and mimicked in compound libraries (e.g. combinatoriallibraries) for screening for microbicidal and/or antiviral activity.

Pharmaceutical Compositions

The invention provides a pharmaceutical composition comprising (a)polypeptide, peptidomimetic, nucleic acid and/or antibody of theinvention and (b) a pharmaceutical carrier.

Component (a) is the active ingredient in the composition, and this ispresent at a therapeutically effective amount i.e. an amount sufficientto inhibit microbial/viral growth and/or survival in a patient, andpreferably an amount sufficient to eliminate microbial infection. Theprecise effective amount for a given patient will depend upon their sizeand health, the nature and extent of infection, and the composition orcombination of compositions selected for administration. The effectiveamount can be determined by routine experimentation and is within thejudgment of the clinician. For purposes of the present invention, aneffective dose will generally be from about 0.01 mg/kg to about 5 mg/kg,or about 0.01 mg/ kg to about 50 mg/kg or about 0.05 mg/kg to about 10mg/kg. Pharmaceutical compositions based on polypeptides, antibodies andnucleic acids are well known in the art. Polypeptides may be included inthe composition in the form of salts and/or esters.

Carrier (b) can be any substance that does not itself induce theproduction of antibodies harmful to the patient receiving thecomposition, and which can be administered without undue toxicity.Suitable carriers can be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Pharmaceutically acceptable carriers can include liquids suchas water, saline, glycerol and ethanol. Auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,can also be present in such vehicles. Liposomes are suitable carriers. Athorough discussion of pharmaceutical carriers is available in ref. 68.

Viral and microbial infections affect various areas of the body and sothe compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared. The composition may be prepared for topical administratione.g. as an ointment, cream or powder. The composition be prepared fororal administration e.g. as a tablet or capsule, or as a syrup(optionally flavoured). The composition may be prepared for pulmonaryadministration e.g. as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g. as drops,as a spray, or as a powder [e.g. 69]. The composition may be included ina mouthwash. The composition may be lyophilised.

The pharmaceutical composition is preferably sterile. It is preferablypyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8,generally around pH 7.

The invention also provides a delivery device containing apharmaceutical composition of the invention. The device may be, forexample, a syringe or an inhaler.

Compositions of the invention may be used in conjunction with knownanti-fungals. Suitable anti-fungals include, but are not limited to,azoles (e.g. fluconazole, itraconazole), polyenes (e.g. amphotericin B),flucytosine, and squalene epoxidase inhibitors (e.g. terbinafine) [seealso ref. 70]. Compositions may also be used in conjunction with knownantivirals e.g. HIV protease inhibitors, a 2′,3′-dideoxynucleoside (e.g.DDC, DDI), 3′-azido-2′,3′-dideoxynucleosides (AZT),3′-fluoro-2′,3′-dideoxynucleosides (FLT),2′,3′-didehydro-2′,3′-dideoxynucleosides (e.g. D4C, D4T) and carbocyclicderivatives thereof (e.g. carbovir),2′-fluoro-ara-2′,3′-dideoxynucleosides, 1,3-dioxolane derivatives (e.g.2′,3′-dideoxyl-3′-thiacytidine), oxetanocin analogues and carbocyclicderivatives thereof (e.g. cyclobut-G) and the9-(2-phosphonylmethoxyethyl)adenine (PMEA) and9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) derivatives,tetrahydro-imidazo[4,5,l -jk][1,4]-benzodiazepin-2(1H)one (TIBO),1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine (HEPT),dipyrido[3,2-b:2′,3′-e]-[1,4]diazepin-6-one (nevirapine) andpyridin-2(1H)one derivatives, 3TC, etc.

Medical Treatments and Uses

The invention provides antibody, polypeptide, peptidomimetic or nucleicacid of the invention for use as a medicament. The invention alsoprovides a method for treating a patient suffering from a microbialand/or viral infection, comprising administering to the patient apharmaceutical composition of the invention. The invention also providesthe use of antibody, polypeptide, peptidomimetic or nucleic acid of theinvention in the manufacture of a medicament for treating a patient.

The patient is preferably a human. The human may be an adult or,preferably, a child. The human may be immunocompromised.

These uses and methods are particularly useful for treating infectionsof: Candida species, such as C.albicans; Cryptococcus species, such asC.neoformans; Enterococcus species, such as E.faecalis; Streptococcusspecies, such as S.pneumoniae, S.mutans, S.agalactiae and S.pyogenes;Leishmania species, such as L.major and L.infantum; Acanthamoebaspecies, such as A.castellani; Aspergillus species, such as A.fumigatusand A.flavus; Pneumocystis species, such as P.carinii; Mycobacteriumspecies, such as M.tuberculosis; Pseudomonas species, such asP.aeruginosa; Staphylococcus species, such as S.aureus; Salmonellaspecies, such as S.typhimurium; Coccidioides species such as C.immitis;Trichophyton species such as Tverrucosum; Blastomyces species such asB.dermatidis; Histoplasma species such as H.capsulatum; Paracoccidioidesspecies such as P.brasiliensis; Pythiumn species such as P.insidiosum;and Escherichia species, such as E.coli. They are also useful fortreating infections of: influenza viruses and HIV.

The uses and methods are particularly useful for treating diseasesincluding, but not limited to: candidosis, aspergillosis,cryptococcosis, dermatomycoses, sporothrychosis and other subcutaneousmycoses, blastomycosis, histoplasmosis, coccidiomycosis,paracoccidiomycosis, pneumocystosis, thrush, tuberculosis,mycobacteriosis, respiratory infections, scarlet fever, pneumonia,impetigo, rheumatic fever, sepsis, septicaemia, cutaneous and visceralleishmaniasis, comeal acanthamoebiasis, keratitis, cystic fibrosis,typhoid fever, gastroenteritis and hemolytic-uremic syndrome, flu andAIDS. Anti-C.albicans activity is particularly useful for treatinginfections in AIDS patients.

Efficacy of treatment can be tested by monitoring microbial/viralinfection after administration of the pharmaceutical composition of theinvention.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, ocular, nasal, aural, or pulmonary administration.Injection or intranasal administration is preferred.

Dosage treatment can be a single dose schedule or a multiple doseschedule.

Pharmaceutical compositions of the invention may also be usedprophylactically e.g. in a situation where contact with microbes isexpected and where establishment of infection is to be prevented. Forinstance, the composition may be administered prior to surgery.

Polypeptide Expression and Other General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature e.g. SambrookMolecular Cloning; A Laboratory Manual, Second Edition (1989); DNACloning, Volumes I and ii (D. N Glover ed. 1985); OligonucleolideSynthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames &S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide to Molecular Cloning (1984); the Methods in Enzymology series(Academic Press, Inc.), especially volumes 154 & 155; Gene TransferVectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987),Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); Scopes, (1987) Protein Purification: Principles and Practice,Second Edition (Springer-Verlag, N.Y.), and Handbook of ExperimentalImmunology, Volumes I-IV (Weir& Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in thisspecification: Nucleotides A Adenine C Cytosine G Guanine T Thymine UUracil

Degenerate nucleotide codes in addition to the five above codes: N any RpuRine Y pYrimidine K Keto (A/C/G/T) (G/A) (T/C) (G/T) M aMino S StrongW Weak B not A (A/C) (G/C) (A/T) (C/G/T) D not C H not G V not T (A/G/T)(A/C/T) (A/C/G)

Amino acids A Alanine C Cysteine D Aspartate E Glutamate F Phenyl- GGlycine H Histidine I Isoleucine alanine L Leucine M Methionine NAsparagine K Lysine Q Glutamine R Arginine S Serine P Proline V Valine WTryptophan Y Tyrosine T ThreonineThe invention provides a polypeptide or nucleic acid comprising orconsisting of any one of amino acid or nucleotide sequences given in thesequence listing.

Definitions

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional to X, such as X+Y.

A composition containing X is “substantially free of” Y when at least85% by weight of the total X+Y in the composition is X. Preferably, Xcomprises at least about 90% by weight of the total of X+Y in thecomposition, more preferably at least about 95% or even 99% by weight.The term “heterologous” refers to two biological components that are notfound together in nature. The components may be host cells, genes, orregulatory regions, such as promoters. Although the heterologouscomponents are not found together in nature, they can function together,as when a promoter heterologous to a gene is operably linked to thegene. Another example is where an influenza sequence is heterologous toa mouse host cell. Further examples would be two epitopes from the sameor different proteins which have been assembled in a single protein inan arrangement not found in nature.

An “origin of replication” is a polynucleotide sequence that initiatesand regulates replication of polynucleotides, such as an expressionvector. The origin of replication behaves as an autonomous unit ofpolynucleotide replication within a cell, capable of replication underits own control. An origin of replication may be needed for a vector toreplicate in a particular host cell. With certain origins ofreplication, an expression vector can be reproduced at a high copynumber in the presence of the appropriate proteins within the cell.Examples of origins are the autonomously replicating sequences, whichare effective in yeast; and the viral T-antigen, effective in COS-7cells. A “mutant” sequence is defined as DNA, RNA or amino acid sequencediffering from but having sequence identity with the native or disclosedsequence. Depending on the particular sequence, the degree of sequenceidentity between the native or disclosed sequence and the mutantsequence is preferably greater than 50% (e.g. 60%, 70%, 80%, 90%, 95%,99% or more, calculated using the Smith-Waterman algorithm as describedabove). As used herein, an “allelic variant” of a nucleic acid molecule,or region, for which nucleic acid sequence is provided herein is anucleic acid molecule, or region, that occurs essentially at the samelocus in the genome of another or second isolate, and that, due tonatural variation caused by, for example, mutation or recombination, hasa similar but not identical nucleic acid sequence. A coding regionallelic variant typically encodes a protein having similar activity tothat of the protein encoded by the gene to which it is being compared.An allelic variant can also comprise an alteration in the 5′ or 3′untranslated regions of the gene, such as in regulatory control regions(e.g. see U.S. Pat. No. 5,753,235).

Polypeptide Expression

Nucleotide sequences can be expressed in a variety of differentexpression systems; for example those used with mammalian cells,baculoviruses, plants, bacteria, and yeast.

Generally, any system or vector that is suitable to maintain, propagateor express nucleic acid molecules to produce a polypeptide in therequired host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those described in Sambrooket. al. Generally, the encoding gene can be placed under the control ofa control element such as a promoter, ribosome binding site (forbacterial expression) and, optionally, an operator, so that the DNAsequence encoding the desired polypeptide is transcribed into RNA in thetransformed host cell.

Examples of suitable expression systems include, for example,chromosomal, episomal and virus-derived systems, including, for example,vectors derived from: bacterial plasmids, bacteriophage, transposons,yeast episomes, insertion elements, yeast chromosomal elements, virusessuch as baculoviruses, papova viruses such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,or combinations thereof, such as those derived from plasmid andbacteriophage genetic elements, including cosmids and phagemids. Humanartificial chromosomes (HACs) may also be employed to deliver largerfragments of DNA than can be contained and expressed in a plasmid.

Particularly suitable expression systems include microorganisms such asbacteria transformed with recombinant bacteriophage, plasmid or cosmidDNA expression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (for example,baculovirus); plant cell systems transformed with virus expressionvectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or with bacterial expression vectors (for example, Ti orpBR322 plasmids); or animal cell systems. Cell-free translation systemscan also be employed to produce the polypeptides of the invention.

Introduction of nucleic acid molecules encoding a polypeptide of thepresent invention into host cells can be effected by methods describedin many standard laboratory manuals, such as Davis et al., Basic Methodsin Molecular Biology (1986) and Sambrook et al., (supra). Particularlysuitable methods include calcium phosphate transfection, DEAE-dextranmediated transfection, transvection, microinjection, cationiclipid-mediated transfection, electroporation, transduction, scrapeloading, ballistic introduction or infection (see Sambrook et al., 1989[supra]; “Current Protocols in Molecular Biology”, Ausubel et al. (eds).Greene Publishing Association and John Wiley lnterscience, New York,1989, 1992; Spector, Goldman & Leinwald, 1998). In eukaryotic cells,expression systems may either be transient (for example, episomal) orpermanent (chromosomal integration) according to the needs of thesystem.

For long-term, high-yield production of a recombinant polypeptide,stable expression is preferred. For example, cell lines which stablyexpress the polypeptide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may bcallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells that successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

i. Mammalian Systems

Mammalian expression systems are known in the art. A mammalian promoteris any DNA sequence capable of binding mammalian RNA polymerase andinitiating the downstream (3′) transcription of a coding sequence (e.g.structural gene) into mRNA. A promoter will have a transcriptioninitiating region, which is usually placed proximal to the 5′ end of thecoding sequence, and a TATA box, usually located 25-30 base pairs (bp)upstream of the transcription initiation site. The TATA box is thoughtto direct RNA polymerase II to begin RNA synthesis at the correct site.A mammalian promoter will also contain an upstream promoter element,usually located within 100 to 200 bp upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation [Sambrook et al. (1989)“Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: ALaboratory Manual, 2nd ed.].

Mammalian viral genes are often highly expressed and have a broad hostrange; therefore sequences encoding mammalian viral genes provideparticularly useful promoter sequences. Examples include the SV40 earlypromoter, mouse mammary tumor virus LTR promoter, adenovirus major latepromoter (Ad MLP), and herpes simplex virus promoter. In addition,sequences derived from non-viral genes, such as the murinemetallotheionein gene, also provide useful promoter sequences.Expression may be either constitutive or regulated (inducible),depending on the promoter can be induced with glucocorticoid inhormone-responsive cells.

The presence of an enhancer element (enhancer), combined with thepromoter elements described above, will usually increase expressionlevels. An enhancer is a regulatory DNA sequence that can stimulatetranscription up to 1000-fold when linked to homologous or heterologouspromoters, with synthesis beginning at the normal RNA start site.Enhancers are also active when they are placed upstream or downstreamfrom the transcription initiation site, in either normal or flippedorientation, or at a distance of more than 1000 nucleotides from thepromoter [Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989)Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived fromviruses may be particularly useful, because they usually have a broaderhost range. Examples include the SV40 early gene enhancer [Dijkema et al(1985) EMBO J. 4:761 ] and the enhancer/promoters derived from the longterminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b)PNAS USA 79:6777] and from human cytomegalovirus [Boshart et al. (1985)Cell 41:521]. Additionally, some enhancers are regulatable and becomeactive only in the presence of an inducer, such as a hormone or metalion [Sassone-Corsi and Borelli (1986) Trends Genel. 2:215; Maniatis etal. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. Apromoter sequence may be directly linked with the DNA molecule, in whichcase the first amino acid at the N-terminus of the recombinant proteinwill always be a methionine, which is encoded by the ATG start codon. Ifdesired, the N-terminus may be cleaved by in vitro incubation withcyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provides forsecretion of the foreign protein in mammalian cells. Preferably, thereare processing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell. Theadenovirus triparite leader is an example of a leader sequence thatprovides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-transcriptional cleavage and polyadenylation[Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988)“Termination and 3′ end processing of eukaryotic RNA. In Transcriptionand splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) TrendsBiochem. Sci. 14:105]. These sequences direct the transcription of anmRNA which can be translated into the polypeptide encoded by the DNA.Examples of transcription terminater/polyadenylation signals includethose derived from SV40 [Sambrook et al (1989) “Expression of clonedgenes in cultured mammalian cells.” In Molecular Cloning: A LaboratoryManual].

Usually, the above described components, comprising a promoter,polyadenylation signal, and transcription termination sequence are puttogether into expression constructs. Enhancers, introns with functionalsplice donor and acceptor sites, and leader sequences may also beincluded in an expression construct, if desired. Expression constructsare often maintained in a replicon, such as an extrachromosomal element(e.g. plasmids) capable of stable maintenance in a host, such asmammalian cells or bacteria. Mammalian replication systems include thosederived from animal viruses, which require trans-acting factors toreplicate. For example, plasmids containing the replication systems ofpapovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] orpolyomavirus, replicate to extremely high copy number in the presence ofthe appropriate viral T antigen. Additional examples of mammalianreplicons include those derived from bovine papillomavirus andEpstein-Barr virus. Additionally, the replicon may have two replicatonsystems, thus allowing it to be maintained, for example, in mammaliancells for expression and in a prokaryotic host for cloning andamplification. Examples of such mammalian-bacteria shuttle vectorsinclude pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO[Shimizu et al. (1986) Mol Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to betransformed. Methods for introduction of heterologous polynucleotidesinto mammalian cells are known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalised cell lines available from the AmericanType Culture Collection (ATCC) including, but not limited to, Chinesehamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney(COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellularcarcinoma (for example Hep G2) cells and a number of other cell lines.

ii. Baculovirus Systems

The polynucleotide encoding the protein can also be inserted into asuitable insect expression vector, and is operably linked to the controlelements within that vector. Vector construction employs techniqueswhich are known in the art. Generally, the components of the expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene or genes to beexpressed; a wild type baculovirus with a sequence homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfervector, the vector and the wild type viral genome are transfected intoan insect host cell where the vector and viral genome are allowed torecombine. The packaged recombinant virus is expressed and recombinantplaques are identified and purified. Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form e.g. from Invitrogen, San Diego Calif. (“MaxBac” kit). Thesetechniques are generally known to those skilled in the art and fullydescribed in Summers & Smith, Texas Agricultural Experiment StationBulletin No. 1555 (1987) (hereinafter “Summers & Smith”).

Prior to inserting the DNA sequence encoding the protein into thebaculovirus genome, the above described components, comprising apromoter, leader (if desired), coding sequence of interest, andtranscription termination sequence, are usually assembled into anintermediate transplacement construct (transfer vector). This constructmay contain a single gene and operably linked regulatory elements;multiple genes, each with its owned set of operably linked regulatoryelements; or multiple genes, regulated by the same set of regulatoryelements. Intermediate transplacement constructs are often maintained ina replicon, such as an extrachromosomal element (e.g. plasmids) capableof stable maintenance in a host, such as a bacterium. The replicon willhave a replication system, thus allowing it to be maintained in asuitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducingforeign genes into AcNPV is pAc373. Many other vectors, known to thoseof skill in the art, have also been designed. These include, forexample, pVL985 (which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 basepairs downstreamfrom the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains a polyhedrin polyadenylation signal(Miller (1988) Ann. Rev. Microbiol. 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E.coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (5′ to 3′)transcription of a coding sequence (e.g. structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region usually includes an RNA polymerase binding site and atranscription initiation site. A baculovirus transfer vector may alsohave a second domain called an enhancer, which, if present, is usuallydistal to the structural gene. Expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in a viralinfection cycle, provide particularly useful promoter sequences.Examples include sequences derived from the gene encoding the viralpolyhedron protein, Friesen et al., (1986) “The Regulation ofBaculovirus Gene Expression,” in The Molecular Biology of Baculoviruses(ed. Walter Doerfler); EP-127839 & EP-155476; and the gene encoding thep10 protein, Vlak et al. (1988), J Gen. ViroL 69:765.

DNA encoding suitable signal sequences can be derived from genes forsecreted insect or baculovirus proteins, such as the baculoviruspolyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively,since the signals for mammalian cell posttranslational modifications(such as signal peptide cleavage, proteolytic cleavage, andphosphorylation) appear to be recognized by insect cells, and thesignals required for secretion and nuclear accumulation also appear tobe conserved between the invertebrate cells and vertebrate cells,leaders of non-insect origin, such as those derived from genes encodinghuman □-inteferon, Maeda et al., (1985), Nature 315:592; humangastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. CellBiol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad Sci.USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; andhuman glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also beused to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressedintracellularly or, if it is expressed with the proper regulatorysequences, it can be secreted. Good intracellular expression of nonfusedforeign proteins usually requires heterologous genes that ideally have ashort leader sequence containing suitable translation initiation signalspreceding an ATG start signal. If desired, methionine at the N-terminusmay be cleaved from the mature protein by in vitro incubation withcyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are notnaturally secreted can be secreted from the insect cell by creatingchimeric DNA molecules that encode a fusion protein comprised of aleader sequence fragment that provides for secretion of the foreignprotein in insects. The leader sequence fragment usually encodes asignal peptide comprised of hydrophobic amino acids which direct thetranslocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding theexpression product precursor of the protein, an insect cell host isco-transformed with the heterologous DNA of the transfer vector and thegenomic DNA of wild type baculovirus—usually by co-transfection. Thepromoter and transcription termination sequence of the construct willusually comprise a 2-5kb section of the baculovirus genome. Methods forintroducing heterologous DNA into the desired site in the baculovirusvirus are known in the art. (See Summers & Smith supra; Ju et al.(1987); Smith et al., Mol. Cell Biol. (1983) 3:2156; and Luckow andSummers (1989)). For example, the insertion can be into a gene such asthe polyhedrin gene, by homologous double crossover recombination;insertion can also be into a restriction enzyme site engineered into thedesired baculovirus gene. Miller et al., (1989), Bioessoys 4:91.The DNAsequence, when cloned in place of the polyhedrin gene in the expressionvector, is flanked both 5′ and 3′ by polyhedrin-specific sequences andis positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packagedinto an infectious recombinant baculovirus. Homologous recombinationoccurs at low frequency (between about 1% and about 5%); thus, themajority of the virus produced after cotransfection is still wild-typevirus. Therefore, a method is necessary to identify recombinant viruses.An advantage of the expression system is a visual screen allowingrecombinant viruses to be distinguished. The polyhedrin protein, whichis produced by the native virus, is produced at very high levels in thenuclei of infected cells at late times after viral infection.Accumulated polyhedrin protein forms occlusion bodies that also containembedded particles. These occlusion bodies, up to 15 □m in size, arehighly refractile, giving them a bright shiny appearance that is readilyvisualized under the light microscope. Cells infected with recombinantviruses lack occlusion bodies. To distinguish recombinant virus fromwild-type virus, the transfection supernatant is plaqued onto amonolayer of insect cells by techniques known to those skilled in theart. Namely, the plaques are screened under the light microscope for thepresence (indicative of wild-type virus) or absence (indicative ofrecombinant virus) of occlusion bodies. Current Protocols inMicrobiology Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10,1990);Summers & Smith, supra; Miller et al. (1989). Recombinant baculovirusexpression vectors have been developed for infection into several insectcells. For example, recombinant baculoviruses have been developed for,inter alia: Aedes aegypli, Aulographa californica, Bombyx mori,Drosophila melanogaster, Spodoplerafrugiperda, and Trichoplusia ni (WO89/046699; Carbonell et al., (1985) J ViroL 56:153; Wright (1986) Nature321:718; Smith et al., (1983) Mol. CelL BioL 3:2156; and see generally,Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both directand fusion expression of heterologous polypeptides in abaculovirus/expression system; cell culture technology is generallyknown to those skilled in the art. See, e.g. Summers & Smith supra.

The modified insect cells may then be grown in an appropriate nutrientmedium, which allows for stable maintenance of the plasmid(s) present inthe modified insect host. Where the expression product gene is underinducible control, the host may be grown to high density, and expressioninduced. Alternatively, where expression is constitutive, the productwill be continuously expressed into the medium and the nutrient mediummust be continuously circulated, while removing the product of interestand augmenting depleted nutrients. The product may be purified by suchtechniques as chromatography, e.g. HPLC, affinity chromatography, ionexchange chromatography, etc.; electrophoresis; density gradientcentrifugation; solvent extraction, or the like. As appropriate, theproduct may be further purified, as required, so as to removesubstantially any insect proteins which are also secreted in the mediumor result from lysis of insect cells, so as to provide a product whichis at least substantially free of host debris, e.g. proteins, lipids andpolysaccharides.

In order to obtain protein expression, recombinant host cells derivedfrom the transformants are incubated under conditions which allowexpression of the recombinant protein encoding sequence. Theseconditions will vary, dependent upon the host cell selected. However,the conditions are readily ascertainable to those of ordinary skill inthe art, based upon what is known in the art.

iii. Plant Systems

There are many plant cell culture and whole plant genetic expressionsystems known in the art. Exemplary plant cellular genetic expressionsystems include those described in patents, such as: U.S. Pat. No.5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143.Additional examples of genetic expression in plant cell culture has beendescribed by Zenk, Phylochemistry 30:3861-3863 (1991). Descriptions ofplant protein signal peptides may be found in addition to the referencesdescribed above in Vaulcombe et al., Mol. Gen. Genel. 209:33-40 (1987);Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, JBiol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356(1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987);Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene122:247-253 (1992). A description of the regulation of plant geneexpression by the phytohormone, gibberellic acid and secreted enzymesinduced by gibberellic acid can be found in R. L. Jones and J.MacMillin, Gibberellins: in: Advanced Plant Physiology,. Malcolm B.Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52.References that describe other metabolically-regulated genes: Sheen,Plant Cell, 2:1027-1038(1990); Maas et al., EMBO J. 9:3447-3452 (1990);Benkel & Hickey, PNAS USA 84:1337-1339 (1987) Typically, usingtechniques known in the art, a desired polynucleotide sequence isinserted into an expression cassette comprising genetic regulatoryelements designed for operation in plants. The expression cassette isinserted into a desired expression vector with companion sequencesupstream and downstream from the expression cassette suitable forexpression in a plant host. The companion sequences will be of plasmidor viral origin and provide necessary characteristics to the vector topermit the vectors to move DNA from an original cloning host, such asbacteria, to the desired plant host. The basic bacterial/plant vectorconstruct will preferably provide a broad host range prokaryotereplication origin; a prokaryote selectable marker, and, forAgrobacterium transformations, T DNA sequences forAgrobacterium-mediated transfer to plant chromosomes. Where theheterologous gene is not readily amenable to detection, the constructwill preferably also have a selectable marker gene suitable fordetermining if a plant cell has been transformed. A general review ofsuitable markers, e.g. for the members of the grass family, is found inWilmink & Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

Sequences suitable for permitting integration of the heterologoussequence into the plant genome are also recommended. These might includetransposon sequences and the like for homologous recombination as wellas Ti sequences which permit random insertion of a heterologousexpression cassette into a plant genome. Suitable prokaryote selectablemarkers include resistance toward antibiotics such as ampicillin ortetracycline. Other DNA sequences encoding additional functions may alsobe present in the vector, as is known in the art.

The nucleic acid molecules of the subject invention may be included intoan expression cassette for expression of the protein(s) of interest.Usually, there will be only one expression cassette, although two ormore are feasible. The recombinant expression cassette will contain inaddition to the heterologous protein encoding sequence the followingelements, a promoter region, plant 5′ untranslated sequences, initiationcodon depending upon whether or not the structural gene comes equippedwith one, and a transcription and translation termination sequence.Unique restriction enzyme sites at the 5′ and 3′ ends of the cassetteallow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to thepresent invention. The sequence encoding the protein of interest willencode a signal peptide which allows processing and translocation of theprotein, as appropriate, and will usually lack any sequence which mightresult in the binding of the desired protein of the invention to amembrane. Since, for the most part, the transcriptional initiationregion will be for a gene which is expressed and translocated duringgermination, by employing the signal peptide which provides fortranslocation, one may also provide for translocation of the protein ofinterest. In this way, the protein(s) of interest will be translocatedfrom the cells in which they are expressed and may be efficientlyharvested. Typically secretion in seeds are across the aleurone orscutellar epithelium layer into the endosperm of the seed. While it isnot required that the protein be secreted from the cells in which theprotein is produced, this facilitates the isolation and purification ofthe recombinant protein.

Since the ultimate expression of the desired gene product will be in aeucaryotic cell it is desirable to determine whether any portion of thecloned gene contains sequences which will be processed out as introns bythe host's splicosome machinery. If so, site-directed mutagenesis of the“intron” region may be conducted to prevent losing a portion of thegenetic message as a false intron code, Reed and Maniatis, Cell41:95-105, 1985.

The vector can be microinjected directly into plant cells by use ofmicropipettes to mechanically transfer the recombinant DNA. Crossway,Mol. Gen Genet, 202:179-185, 1985. The genetic material may also betransferred into the plant cell by using polyethylene glycol, Krens, etal., Nature, 296, 72-74, 1982. Another method of introduction of nucleicacid segments is high velocity ballistic penetration by small particleswith the nucleic acid either within the matrix of small beads orparticles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particlebombardment of barley endosperm to create transgenic barley. Yet anothermethod of introduction would be fusion of protoplasts with otherentities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies, Fraley, et al., PNAS USA, 79, 1859-1863, 1982.

The vector may also be introduced into the plant cells byelectroporation. (Fromm et al., PNAS USA 82:5824, 1985). In thistechnique, plant protoplasts are electroporated in the presence ofplasmids containing the gene construct. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to givewhole regenerated plants can be transformed by the present invention sothat whole plants are recovered which contain the transferred gene. Itis known that practically all plants can be regenerated from culturedcells or tissues, including but not limited to all major species ofsugarcane, sugar beet, cotton, fruit and other trees, legumes andvegetables. Some suitable plants include, for example, species from thegenera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella,Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Dalura, Hyoscyamus, Lycopersion,Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus,Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia,Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Solpiglossis,Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts containing copies ofthe heterologous gene is first provided. Callus tissue is formed andshoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced from the protoplastsuspension. These embryos germinate as natural embryos to form plants.The culture media will generally contain various amino acids andhormones, such as auxin and cytokinins. It is also advantageous to addglutamic acid and proline to the medium, especially for such species ascorn and alfalfa. Shoots and roots normally develop simultaneously.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is fully reproducible and repeatable.

In some plant cell culture systems, the desired protein of the inventionmay be excreted or alternatively, the protein may be extracted from thewhole plant. Where the desired protein of the invention is secreted intothe medium, it may be collected. Alternatively, the embryos andembryoless-half seeds or other plant tissue may be mechanicallydisrupted to release any secreted protein between cells and tissues. Themixture may be suspended in a buffer solution to retrieve solubleproteins. Conventional protein isolation and purification methods willbe then used to purify the recombinant protein. Parameters of time,temperature pH, oxygen, and volumes will be adjusted through routinemethods to optimize expression and recovery of heterologous protein.

iv. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterialpromoter is any DNA sequence capable of binding bacterial RNA polymeraseand initiating the downstream (3′) transcription of a coding sequence(e.g. structural gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5′end of thecoding sequence. This transcription initiation region usually includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter may also have a second domain called an operator,that may overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein may bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression may occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation may be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5′) to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in E.coli [Raibaudet al. (1984) Annu. Rev. Genel. 18:173]. Regulated expression maytherefore be either positive or negative, thereby either enhancing orreducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal. (1977) Nature 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al.(1981) Nucl Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 andEP-A-0121775]. The g-lactamase (bla) promoter system [Weissmann (1981)“The cloning of interferon and other mistakes.” In Interferon 3 (ed. I.Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provideuseful promoter sequences.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of a bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. Forexample, the tac promoter is a hybrid trp-lac promoter comprised of bothtrp promoter and lac operon sequences that is regulated by the lacrepressor [Amann et al. (1983) Gene 25:167; de Boer el al. (1983) PNASUSA 80:21]. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al. (1986)J.Mol.Biol. 189:113; Tabor et al. (1985) PNAS USA 82:1074]. In addition,a hybrid promoter can also be comprised of a bacteriophage promoter andan E.coli operator region (EP-A-0267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E.coli the ribosome binding site is called theShine-Dalgamo (SD) sequence and includes an initiation codon (ATG) and asequence 3-9 nucleotides in length located 3-11 nucleotides upstream ofthe initiation codon [Shine et al. (1975) Nature 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome bybase-pairing between the SD sequence and the 3′ end of 16S rRNA [Steitzet al. (1979) “Genetic signals and nucleotide sequences in messengerRNA.” In Biological Regulation and Development: Gene Expression (ed.R.F. Goldberger)]. To express eukaryotic genes and prokaryotic geneswith weak ribosome-binding site [Sambrook et a. (1989) “Expression ofcloned genes in Escherichia coli.” In Molecular Cloning: A LaboratoryManual].

A DNA molecule may be expressed intracellularly. A promoter sequence maybe directly linked with the DNA molecule, in which case the first aminoacid at the N-terminus will always be a methionine, which is encoded bythe ATG start codon. If desired, methionine at the N-terminus may becleaved from the protein by in vitro incubation with cyanogen bromide orby either in vivo on in vitro incubation with a bacterial methionineN-terminal peptidase (EPO-A-0 219 237).

Fusion proteins provide an alternative to direct expression. Usually, aDNA sequence encoding the N-terminal portion of an endogenous bacterialprotein, or other stable protein, is fused to the 5′ end of heterologouscoding sequences. Upon expression, this construct will provide a fusionof the two amino acid sequences. For example, the bacteriophage lambdacell gene can be linked at the 5′terminus of a foreign gene andexpressed in bacteria. The resulting fusion protein preferably retains asite for a processing enzyme (factor Xa) to cleave the bacteriophageprotein from the foreign gene [Nagai et al. (1984) Nature 309:810].Fusion proteins can also be made with sequences from the lacZ [Jia etal. (1987) Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93;Makoffet et al. (1989) J. Gen Microbiol. 135:11], and Chey [EP-A-0 324647] genes. The DNA sequence at the junction of the two amino acidsequences may or may not encode a cleavable site. Another example is aubiquitin fusion protein. Such a fusion protein is made with theubiquitin region that preferably retains a site for a processing enzyme(e.g. ubiquitin specific processing-protease) to cleave the ubiquitinfrom the foreign protein. Through this method, native foreign proteincan be isolated [Miller et al. (1989) Bio/Technology 7:698].

Alternatively, foreign proteins can also be secreted from the cell bycreating chimeric DNA molecules that encode a fusion protein comprisedof a signal peptide sequence fragment that provides for secretion of theforeign protein in bacteria [U.S. Pat. No. 4,336,336]. The signalsequence fragment usually encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic space, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).Preferably there are processing sites, which can be cleaved either invivo or in vitro encoded between the signal peptide fragment and theforeign gene.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as E.coli outer membrane protein gene(ompA) [Masui et al. (1983) in: Experimental Manipulation of GeneExpression; Ghrayeb et al. (1984) EMBO J 3:2437] and the E.coli alkalinephosphatase signal sequence (phoA) [Oka et al. (1985) PNAS USA 82:7212].As a further example, signal sequence of the alpha-amylase gene fromvarious Bacillus strains can be used to secrete heterologous proteinsfrom B.subtilis [Palva et al. (1982) PNAS USA 79:5582; EP-A-02440421].

Usually, transcription termination sequences recognized by bacteria areregulatory regions located 3′ to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Transcription termination sequencesfrequently include DNA sequences of about 50 nucleotides capable offorming stem loop structures that aid in terminating transcription.Examples include transcription termination sequences derived from geneswith strong promoters, such as the trp gene in E.coli as well as otherbiosynthetic genes.

Usually, the above described components, comprising a promoter, signalsequence (if desired), coding sequence of interest, and transcriptiontermination sequence, are put together into expression constructs.Expression constructs are often maintained in a replicon, such as anextrachromosomal element (e.g. plasmids) capable of stable maintenancein a host, such as bacteria. The replicon will have a replicationsystem, thus allowing it to be maintained in a prokaryotic host eitherfor expression or for cloning and amplification. In addition, a repliconmay be either a high or low copy number plasmid. A high copy numberplasmid will generally have a copy number ranging from ˜5 to ˜200, andusually ˜10 to ˜150. A host containing a high copy number plasmid willpreferably contain at least ˜10, and more preferably at least ˜20plasmids. Either a high or low copy number vector may be selected,depending upon the effect of the vector and the foreign protein on thehost.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to the bacterial chromosomethat allows the vector to integrate. Integrations appear to result fromrecombinations between homologous DNA in the vector and the bacterialchromosome. For example, integrating vectors constructed with DNA fromvarious Bacillus strains integrate into the Bacillus chromosome (EP-A-0127328). Integrating vectors may also be comprised of bacteriophage ortransposon sequences.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of bacterialstrains that have been transformed. Selectable markers can be expressedin the bacterial host and may include genes which render bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev.Microbiol. 32:469]. Selectable markers may also include biosyntheticgenes, such as those in the histidine, tryptophan, and leucinebiosynthetic pathways.

Alternatively, some of the above described components can be puttogether in transformation vectors. Transformation vectors are usuallycomprised of a selectable market that is either maintained in a repliconor developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomalreplicons or integrating vectors, have been developed for transformationinto many bacteria. For example, expression vectors have been developedfor, inter alia, the following bacteria: Bacillus subtilis [Palva et.al. (1982) PNAS USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128;Amann el al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol.189:113; EP-A-0 036 776,EP-A-0 136 829 and EP-A-0 136 907],Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbial.54:655]; Streptococcus lividans [Powell el al. (1988) Appl. EnvironMicrobial. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-knownin the art, and usually include either the transformation of bacteriatreated with CaCl₂ or other agents, such as divalent cations and DMSO.DNA can also be introduced into bacterial cells by electroporation.Transformation procedures usually vary with the bacterial species to betransformed. See e.g. [Masson et al. (1989) FEMS Microbiol. Lett.60:273; Palva et al. (1982) PNAS USA 79:5582; EP-A-0 036 259 and EP-A-0063 953; WO 84/04541, Bacillus], [Miller et al. (1988) PNAS USA 85:856;Wang el al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al.(1973) PNAS USA 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127;Kushner (1978) “An improved method for transformation of Escherichiacoli with ColE1-derived plasmids. In Genetic Engineering: Proceedings ofthe International Symposium on Genetic Engineering (eds. H.W. Boyer andS. Nicosia); Mandel et al. (1970)J. Mol. Biol. 53:159; Taketo (1988)Biochim. Biophys. Acta 949:318; Escherichia], [Chassy el al. (1987) FEMSMicrobioL Lett. 44:173 Lactobacillus]; [Fiedler et. al. (1988) AnaLBiochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS MicrobioLLett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol.144:698; Hariander (1987) “Transformation of Streptococcus lactis byelectroporation, in: Streptococcal Genetics (ed. J. Ferretti and R.Curtiss III); Perry et al. (1981) Infect. Immun 32:1295; Powellet et al.(1988) Appl. Environ. Microbiol. 54:655; Somkutiet al. (1987) Proc. 4thEvr. Cong. Biotechnology 1:412, Streptococcus].

General guidance on expression in E.coli and its optimisation can befound in Baneyx (1999) Curr.Opin.Biolech. 10:411421 and Hannig &Makrides (1998) TIBTECH 16:54-60.

v. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in theart. A yeast promoter is any DNA sequence capable of binding yeast RNApolymerase and initiating the downstream (3′) transcription of a codingsequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regionusually includes an RNA polymerase binding site (the “TATA Box”) and atranscription initiation site. A yeast promoter may also have a seconddomain called an upstream activator sequence (UAS), which, if present,is usually distal to the structural gene. The UAS permits regulated(inducible) expression. Constitutive expression occurs in the absence ofa UAS. Regulated expression may be either positive or negative, therebyeither enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway,therefore sequences encoding enzymes in the metabolic pathway provideparticularly useful promoter sequences. Examples include alcoholdehydrogenase (ADH) (EP-A-0284044), glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, enolase, glucokinase,and pyruvate kinase (PyK) (EPO-A-0329203). The yeast PHO5 gene, encodingacid phosphatase, also provides useful promoter sequences [Myanohara etal. (1983) PNAS USA 80:1].

In addition, synthetic promoters which do not occur in nature alsofunction as yeast promoters. For example, UAS sequences of one yeastpromoter may be joined with the transcription activation region ofanother yeast promoter, creating a synthetic hybrid promoter. Examplesof such hybrid promoters include the ADH regulatory sequence linked tothe GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and4,880,734). Other examples of hybrid promoters include promoters whichconsist of the regulatory sequences of either the ADH2, GAL4, GAL10, ORPHO5 genes, combined with the transcriptional activation region of aglycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore,a yeast promoter can include naturally occurring promoters of non-yeastorigin that have the ability to bind yeast RNA polymerase and initiatetranscription. Examples of such promoters include, inter alia, [Cohen etal. (1980)PNAS USA 77:1078; Henikoff et al. (1981) Nature 283:835;Hollenberg et al. (1981) Curr. Topics MicrobioL Immunol. 96:119;Hollenberg et al. (1979) “The Expression of Bacterial AntibioticResistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids ofMedical, Environmental and Commercial Importance (eds. K.N. Timmis andA. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al.(1980) Curr. Genel 2:109;].

A DNA molecule may be expressed intracellularly in yeast. A promotersequence may be directly linked with the DNA molecule, in which case thefirst amino acid at the N-terminus of the recombinant protein willalways be a methionine, which is encoded by the ATG start codon. Ifdesired, methionine at the N-terminus may be cleaved from the protein byin vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, aswell as in mammalian, baculovirus, and bacterial expression systems.Usually, a DNA sequence encoding the N-terminal portion of an endogenousyeast protein, or other stable protein, is fused to the 5′ end ofheterologous coding sequences. Upon expression, this construct willprovide a fusion of the two amino acid sequences. For example, the yeastor human superoxide dismutase (SOD) gene, can be linked at the 5′terminus of a foreign gene and expressed in yeast. The DNA sequence atthe junction of the two amino acid sequences may or may not encode acleavable site. See e.g. EP-A-0 196 056. Another example is a ubiquitinfusion protein. Such a fusion protein is made with the ubiquitin regionthat preferably retains a site for a processing enzyme (e.g.ubiquitin-specific processing protease) to cleave the ubiquitin from theforeign protein. Through this method, therefore, native foreign proteincan be isolated (e.g. WO88/024066).

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provide forsecretion in yeast of the foreign protein. Preferably, there areprocessing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes forsecreted yeast proteins, such as the invertase gene (EP-A-0 012 873;JPO. 62,096,086) and the A-factor gene (U.S. Pat. No. 4,588,684).Alternatively, leaders of non-yeast origin, such as an interferonleader, exist that also provide for secretion in yeast (EP-A-0 060 057).

A preferred class of secretion leaders are those that employ a fragmentof the yeast alpha-factor gene, which contains both a “pre” signalsequence, and a “pro” region. The types of alpha-factor fragments thatcan be employed include the full-length pre-pro alpha factor leader(about 83 aa residues) as well as truncated alpha-factor leaders(usually about 25 to about 50 amino acid residues) (U.S. Pat. No.4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employingan alpha-factor leader fragment that provides for secretion includehybrid alpha-factor leaders made with a presequence of a first yeast,but a pro-region from a second yeast alphafactor. (e.g. see WO89/02463.)

Usually, transcription termination sequences recognized by yeast areregulatory regions located 3′to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Examples of transcription terminatorsequence and other yeast-recognized termination sequences, such as thosecoding for glycolytic enzymes.

Usually, these components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, areput together into expression constructs. Expression constructs are oftenmaintained in a replicon, such as an extrachromosomal element (e.g.plasmids) capable of stable maintenance in a host, such as yeast orbacteria. The replicon may have two replication systems, thus allowingit to be maintained, for example, in yeast for expression and in aprokaryotic host for cloning and amplification. Examples of suchyeast-bacteria shuttle vectors include YEp24 [Botsteinet et al. (1979)Gene 8:17-24], pCl/l [Brake et al. (1984) PNAS USA 81:4642-4646], andYRp17 [Stinchcomb et al. (1982) J. Mol. Biol. 158:157). In addition, areplicon may be either a high or low copy number plasmid. A high copynumber plasmid will generally have a copy number ranging from ˜5 to˜200, and usually ˜10 to ˜150. A host containing a high copy numberplasmid will preferably have at least ˜10, and more preferably at least˜20. Either a high or low copy number vector may be selected, dependingupon the effect of the vector and the foreign protein on the host. Seee.g. Brakeet et al., supra.

Alternatively, the expression constructs can be integrated into theyeast genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. Integrations appear toresult from recombinations between homologous DNA in the vector and theyeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol.101:228-245]. An integrating vector may be directed to a specific locusin yeast by selecting the appropriate homologous sequence for inclusionin the vector. See Orr-Weaver et al., supra. One or more expressionconstruct may integrate, possibly affecting levels of recombinantprotein produced [Rine et al. (1983) PNAS USA 80:6750]. The chromosomalsequences included in the vector can occur either as a single segment inthe vector, which results in the integration of the entire vector, ortwo segments homologous to adjacent segments in the chromosome andflanking the expression construct in the vector, which can result in thestable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of yeast strainsthat have been transformed. Selectable markers may include biosyntheticgenes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2,TRP1, and ALG7, and the G418 resistance gene, which confer resistance inyeast cells to tunicamycin and G418, respectively. In addition, asuitable selectable marker may also provide yeast with the ability togrow in the presence of toxic compounds, such as metal. For example, thepresence of CUP1 allows yeast to grow in the presence of copper ions[Butt et al. (1987) Microbiol, Rev. 51:351].

Alternatively, some of the above described components can be puttogether into transformation vectors. Such vectors usually comprise aselectable marker that is either maintained in a replicon or developedinto an integrating vector as described above. Expression andtransformation vectors, either extrachromosomal replicons or integratingvectors, have been developed for transfomnation into many yeasts. Forexample, expression vectors have been developed for, inter alia, thefollowing yeasts:Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol.6:142], Candida maltosa [Kunze, et al. (1985) J. Basic Microbiol.25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen Microbiol.132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302],Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165],Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol.154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichiaguillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichiapastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos.4,837,148 and 4,929,555,], Saccharomyces cerevisiae [Hinnen et al.(1978) PNAS USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163],Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], andYarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471Gaillardin, et al. (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known inthe art, and usually include either the transformation of spheroplastsor of intact yeast cells treated with alkali cations. Transformationprocedures usually vary with the yeast species to be transformed. Seee.g. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J.Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. GenMicrobiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen Genet. 202:302;Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt etal. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990)Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell.Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat.Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) PNAS USA75;1929; Ito et al. (1983)J. Bacteriol. 153:163 Saccharomyces]; [Beachand Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al.(1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49;Yarrowia].

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows structural and functional relationships of Pichia anomalakiller toxin (PaKT), PaKT-neutralizing monoclonal antibody (mAb KT4),killer toxin receptor (KTR), and PaKT-like killer antibodies andderivative killer mimotopes (KM).

FIG. 2 shows in vitro microbicidal activity by CFU assay of mAb K10 andmAb K20 in comparison with an irrelevant isotype-matched mAb (all at 100μg/ml dose) against Candida albicans UP10S.

FIG. 3 shows in vitro microbicidal activity of scFv antibodies H6 andH20 (100 μg/ml dose).

FIG. 4 shows in vitro microbicidal activity of peptides KM2 and KM3compared to KM0 and IP controls (1 mg/ml dose).

FIG. 5 shows in vitro microbicidal activity of KM4 (0.5 mg/ml dose).

FIG. 6 shows in vitro microbicidal activity of KM5 (1 mg/ml dose).

FIG. 7 shows in vitro microbicidal activity of KM1 (1 mg/ml dose).

FIG. 8 shows clearance of vaginal candidiasis in rats intravaginallyadministered with KM.

FIG. 9 shows a Kaplan-Meyer survival curve of SCID mice challenged with5 LD50 of C. albicans and treated with KM.

FIG. 10 shows microbicidal activity of KM (25 & 10 μg/ml doses) againstC.neoformans UP25.

FIG. 11 shows microbicidal activity of KM (500 μg/ml & 100 μg/ml doses)against S.aureus a38.

FIG. 12 shows in vitro microbicidal activity of KM0, KM6, KM and KM7 at20 μg/ml dose.

FIG. 13 is an illustration of features within the H6 scFv (SEQ ID 2).

FIG. 14 shows the effect of KM peptide (14A & 14B) on A.castellanigrowth, compared to the effect of SP peptide (14C & 14D). Growth was ateither 37° C. (14A & 14C) or 25° C. (14B & 14D). The graphs show thenumber of trophozoites per well.

FIG. 15 shows the effect of KM peptide on A.castellani cell viability.SP peptide is set as 100%.

FIG. 16 shows the effect of the K20 mAb on A.castellani growth, and FIG.17 shows the effect of K10 mAb. Values are the number of trophozoitesper well. Amoebal growth in medium alone is shown as crosses. Antibodieswere used at 12.5 μg/ml (♦), 25μg/ml (Δ) or 50 μg/ml (∘).

FIG. 18 shows the effect of KM (circles) and SP (crosses) peptides oninfluenza virus replication at up to 80 μg/ml concentration. Values arelog₂ HA titres.

FIG. 19 shows the effect of KM (circles) and SP (squares) peptides onHIV-1 replication. Peptides were used at either 1 μg/ml (closed) or 10μg/ml (open). Values are copies/ml over 15 days of culture.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

BRIEF DESCRIPTION OF THE SEQUENCE LISTING SEQ ID Description  1Nucleotide sequence of H6 scFv  2 Amino acid sequence of H6 scFv  3 KM0(fragment of SEQ ID 2)  4 KM (=SEQ ID 3 with Glu→Ala mutation atposition 1)  5-12 Alanine-scanning variants of SEQ ID 3 13 SP0 scramblepeptide 14-20 Shortened derivatives of KM (9mer, 8mer, 7mer, 6mer, 5mer,4mer, 3mer from SEQ ID 3) 21 Nucleotide sequence of H20 scFv 22 Aminoacid sequence of H20 scFv 23 KM1 (=SEQ ID 3 with Cys→Ser mutation atposition 7) 24 KM2 25 KM3 26 KM4 27 KM5 28 Irrelevant peptide IP 29 SP30 Linker (used to join KM0 and KM2 to give KM3) 31 Peptide control 32KM1 derivative (=SEQ ID 4 with Cys→Xaa mutation at position 7) 33 SEQ ID32 with Cys→Ser mutation at position 7 34-38 KM0, KM1, KM2, KM4 & KM5equivalents from H20 39 KM equivalent from H20 40-47 Alanine-scanningvariants of SEQ ID 24 48 KM2 (SEQ ID 24) with Cys→Ser mutation atposition 7 49-57 Alanine-scanning variants of SEQ ID 27 58 KM4 (SEQ ID26) with Cys→Ser mutation at position 7 59 C-terminus ‘E-tag’ from scFvsystem 60-65 CDRs from H6 antibody (fragments of SEQ ID 2) 66-71 CDRsfrom H20 antibody (fragments of SEQ ID 22)NB: the inclusion of a polypeptide sequence in the sequence listing doesnot imply any particular D- or L-configuration to its constituent aminoacids.

MODES FOR CARRYING OUT THE INVENTION

H6 Single-Chain Fv Antibody

H6 is a single-chain Fv raised against the idiotope of the KT4monoclonal antibody. It is an anti-idiotypic antibody raised with thepurpose of mimicking the activity of the Pichia anomala killer toxin(PaKT).

The existence of scFv H6 has previously been reported [e.g. ref. 13],but a method for its manufacture has not previously been disclosed, norhas its amino acid sequence. The sequence of H6 is now disclosed (SEQIDs 1 and 2). Within SEQ ID 2, amino acids 107-132 (GT . . . IE) are alinker and the final 13 amino acids (GA . . . PR; SEQ ID 59) are the‘E-tag’ inserted by the Recombinant Phage Antibody System (PharmaciaBiotech™) used to create the scFv.

The CDRs within H6 are as follows: CDR aa SEQ ID H1 33-37 60 H2 52-65 61H3  98-101 62 L1 153-162 63 L2 178-184 64 L3 217-224 65

The H6 scFv has strong microbicidal effects in vitro against importantpathogenic microorganisms including: C.albicans; C.krusei and C.glabrata (including fluconazole-resistant strains); Cryptococcusneoformans; A. fumigatus; M.tuberculosis; S.aureus, Enterococcusfaecalis, E.faecium, and Streptococcus pneumoniae (includingmethicillin-, vancomycin- and penicillin-resistant strains); S.mutans,Leishmania major, L.infantum and Achantamoeba castellani. Furthermore,it shows specific therapeutic activity in an in vivo model of ratvaginal candidiasis by intravaginal administration.

K20 Monoclonal Antibody and scFv Derivative H20

K10 is an anti-idiotypic rat monoclonal antibody raised against KT4.Like the H6 scFv, it shows good in vitro microbicidal activity. Inaddition, it has been shown to be therapeutic against P.cariniipneumonia in rats infected by aerosol administration [16], and in micetransplanted with T cell depleted bone marrow against aspergillosiscaused by nasal instillation [14].

Anti-idiotypic antibodies were raised in mice against K10 (i.e.anti-anti-anti-idiotypic with respect to PaKT). One of the resultinghybridoma-produced antibodies was designated ‘K20’.

K20 was tested in a conventional in vitro colony forming unit (CFU)assay to evaluate killer activity. Approximately 250 viablePaKT-susceptible germinating C.albicans UP10 cells, suspended in 10 μlPBS, were added to 90 μl K20 to obtain a final concentration of 100μg/ml and incubated for 6 h at 37° C. After incubation with therespective reagents, the fungal cells were dispensed and streaked on thesurface of Sabouraud dextrose agar plates, which were thereforeincubated at 30° C. and their colony forming units (CFU) enumeratedafter 48 hours. Each experiment was performed in triplicate. Anirrelevant mAb was used as a control.

As shown in FIG. 2, K20 shows slightly better anti-candida activity thanK10.

K20 was converted into a scFv antibody using the Recombinant PhageAntibody System (Pharmacia Biotech™). The scFv was designated ‘H20’ andits sequence is given as SEQ IDs 21 and 22. The H20 CDRs are as follows:CDR aa SEQ ID H1 33-37 66 H2 52-66 67 H3  99-115 68 L1 167-176 69 L2192-198 70 L3 231-238 71

H20 was tested in the CFU assay (FIG. 3) and shows a candidacidalactivity comparable to H6.

Active Fragments of H6

Short peptide fragments of H6 were synthesised, with emphasis on the CDRsequences.

Solid phase synthesis was carried out with a MultiSynTech Syro automaticpeptide synthesizer (Witten, Germany) employing Fmoc chemistry with HOBtactivation and Rink amide MBHA resin as solid support. Peptides werecleaved from the resins and deprotected by treatment withtrifluoroacetic acid containing ethandithiol, water, triisobutylsilaneand anisole (93/2.5/2/1.5/1). After precipitation by ethylic ether,peptides were purified by a Vydac C18 column (25 cm x lcm) andcharacterized by amino acid analysis and mass spectrometry. Thefollowing peptides were synthesised: # Name SEQ ID H6 residues Based onCDR 1 KM0 3 146-155 L1 2 KM2 24  91-100 H3 3 KM4 26 146-160 L1 4 KM5 27155-163 L1

These peptides were tested in the CFU assay to evaluate killer activity.

As a control, a ‘scramble’ peptide (SEQ ID 13; SP0’) was synthesised inwhich the amino acids of KM0 were re-ordered to have the same overallpeptide composition but different sequence. This was used in the aboveassay at the same concentration as the test KM peptides as a control forKM0 and KM4. For KM2, the control was SEQ ID 28 (‘IP’ or ‘irrelevantpeptide’ ). For KM5, the control was SEQ ID 31.

Results with KM0 were as follows, expressed as % growth in comparison tothe control: Peptide 100 μg/ml 25 μg/ml 6.25 μg/ml SP0 Control 100 100100 KM0 5.69 ± 0.20 29.84 ± 10.63 67.13 ± 13.81

Results with KM0, KM2, KM4 and KM5 at various concentrations are shownin FIGS. 4 to 6. KM0 & KM5 are thus extremely effective anti-candidalpeptides. KM2 is also effective, and KM4 is moderately effective.

The CDRs of H6 were also prepared in isolated form (SEQ IDs 60-65) andtested for microbicidal activity in the C.albicans CFU assay. Each CDRpeptide was tested at scalar dilutions to establish the IC₅₀. Assayswere performed in triplicate and the IC₅₀of each peptide was calculatedby nonlinear regression analysis of curves obtained by plotting thenumber of CFU versus Log peptide concentration. Results were as follows:Peptide IC₅₀ (mol/l) SEQ ID Heavy chain CDR-1 2.67 × 10⁻⁷ 60 CDR-2 1.17× 10⁻⁷ 61 CDR-3 1.09 × 10⁻⁷ 62 Light chain CDR-1 6.92 × 10⁻⁷ 63 CDR-22.34 × 10⁻⁷ 64 CDR-3 2.00 × 10⁻⁷ 65

The CDRs of H6 antibody therefore show significant anti-candidaactivity, with CDR-3 from the heavy chain (SEQ ID 62) showing the bestactivity.

Fragments of the H6 scFv are therefore able to act as microbicides eventhough they would not be expected to hold in the same tertiaryconformation as in the intact antibody.

Polypeptides Comprising More Than One Fragment of H6

KM0 is derived from a H6 light chain CDR and KM2 is derived from a H6heavy chain CDR. The two decapeptides , each of which has microbididalactivity on its own, were linked by a glycine-rich sequence (SEQ ID 30)to give KM3 (SEQ ID 25).

In comparison to IP in the CFU assay, KM3 showed candidacidal activity,but this was weaker than either KM0 and KM2 alone (FIG. 4).

Substitution within KM0-Cysteine Replacement

The cysteine residue in decapeptide KM0 was substituted with serine togive KM1 (SEQ ID 23), with a view to reducing oxidation andpolymerization processes. KM1 showed similar activity to KM0 in the CFUassay (FIG. 7) in comparison to the IP control. The substitution ofcysteine with serine thus gave no apparent alteration of the antibioticeffect of KM0, but offers increased resistance to oxidation and thusincreased in vivo half life.

SEQ ID 48 is a C→S substitution form of KM2. SEQ ID 58 is a C→Ssubstitution form of KM4.

Substitution Within KM0-Alanine Scanning

Decapeptide KM0 was analysed by alanine scanning [32] in order toidentify the functional contributional of individual residues to itsmicrobicidal activity. Each of the ten constituent amino acids wasreplaced with A (except for residue 9, which is already A) and activityin the in vitro CFU assay was assessed. Results were as follows, withvalues being % growth in comparison to the SP0 control: SEQ ID 100 μg/ml25 μg/ml 6.25 μg/ml 3 5.69 ± 0.20  29.84 ± 10.63  67.13 ± 13.81 4  0  00 5 9.91 ± 3.28 42.67 ± 2.04 53.40 ± 6.99 6 9.27 ± 2.45 19.72 ± 2.9160.06 ± 5.24 7 9.18 ± 4.13 26.56 ± 4.24 63.24 ± 4.77 8 0.10 ± 0.10 10.12± 2.96  40.42 ± 16.01 9 52.89 ± 3.90  55.52 ± 3.7  58.14 ± 8.15 10 55.71± 10.20 59.73 ± 4.77 64.25 ± 6.72 11 2.60 ± 0.50 23.12 ± 3.59 72.95 ±7.42 12 11.90 ± 0.64  32.90 ± 0.79 70.33 ± 9.14 13 100  100  100

The most active peptide is SEQ ID 4 (‘KM’ ), in which the first aminoacid E is substituted by A. SEQ ID 8, in which C was substituted by A,also shows good activity relative to both the SP0 control and to thestarting KM0 decapeptide. The CFU reduction for these two decapeptidescompared to the control was statistically significant at all three doses(p<0.005 by two-tailed Student's t test).

On the basis of KM0 and KM sequences, scramble peptides (SP0, SEQ ID 13;SP, SEQ ID 29) were also synthesised. Neither of these two scramblepeptides showed in vitro candidacidal activity.

Alanine-scanning of KM2 is shown in SEQ IDs 40 to 47.

Alanine-scanning of KM5 is shown in SEQ IDs 49 to 57.

C-Terminal Truncation of KM

SEQ ID 4 (‘KM’) was reduced by C-terminal deletions down to threeresidues to establish the ability to retain in vitro candidacidalactivity relative to the SP control. Scalar dilutions (100-0.8 μg/ml)were tested to establish the minimal fungicidal concentrationcorresponding to the killing of 100% of C. albicans cells. KM and itstrunication derivatives were also tested at scalar dilutions toestablish the peptide concentration (mol/l) corresponding to the 50%inhibitory concentration (IC50). Assays were performed in triplicate andthe IC50 of each peptide was calculated by nonlinear regression analysisof curves obtained by plotting the number of CFU versus Log peptideconcentration using the GraphPad Prism 3.02 software. Results were asfollows: SEQ ID IC50 (mol/l) 4 5.6 × 10⁻⁸ 14   5 × 10⁻⁵ 15 2.3 × 10⁻⁵ 16  6 × 10⁻⁷ 17 7.3 × 10⁻⁴ 18 2.5 × 10⁻⁵ 19 7.1 × 10⁻⁴ 20   1 × 10⁻⁵

There is a drop of candidacidal activity of about three orders ofmagnitude with deletion of the C-terminus serine from KM. However, theheptamer formed by deletion of the 3 C-terminal residues shows activityonly one order of magnitude lower than that of KM.

KM Oligomers

Experiments on the stability of the killer peptide KM afterlyophilisation were carried out using the CFU assay. KM proved to bevery stable in the lyophilised form.

After solubilisation in non-reducing conditions, the free cysteine in KMcan lead to the formation of a disulfide bridge, to give a KM dimer. Thecandidacidal activity of the dimer was assessed by comparison todimerised SP peptide. The disulfide-bonded KM dimer retains candidacidalactivity. Moreover, this activity was maintained unaltered over a longperiod under different storage conditions (4° C. room temperature, 37°C.).

In Vivo Activity of KM in Vaginal Infection Model

KM was tested using a well-established experimental model [71] ofvaginal C. albicans infection in oophorectomized estrogen-treated rats.Estrogen-conditioned rats (5 animals per group) were inoculatedintravaginally with 10⁷ cells of fluconazole-sensitive (SA-40) orfluconazole-resistant (AIDS 68 ) C.albicans. Both Candida strains wereoriginally isolated from human vaginal infection, and maintained instock in the Department of Bacteriology and Medical Mycology of theInstituto Superiore di Sanità (ISS), Rome (Italy). Different doses (10,25, 50 and 100 μg) of KM were administered intravaginally at 1, 24 and48 hours post challenge and vaginal C.albicans burden was quantitated byCFU enumeration from the vaginal fluid taken each day by a specialcalibrated loop. Vaginal smears were also stained by PAS-van Giesonmethod. Any benefit of KM treatment was assessed in terms of microscopicreduction of the hyphal growth in the vagina. Negative controls wereuntreated rats and rats treated with SP. As a positive control, ratsreceived 50 or 100 μg/ml fluconazole (Pfizer) in PBS (0.1 ml ) at 1, 24and 48 hours after the yeast challenge.

A dose-response therapeutic effect was observed at 50 and 100 μg doses.

In subsequent experiments, 50 μg of KM was used in the established threedose administration schedule to determine acceleration of fungal CFUclearance over a period of 28 days. Rats (five per groups) were given10⁷ cells in 0.1 ml of physiological solution on day 0 and were sampledfor initial intravaginal CFU. Treatments were administered 1, 24 and 48hours after the challenge.

FIG. 8 shows the results of a typical experiment with strain SA-40. Ateach time point there was a statistically-significant difference (p<0.05by two-tailed Student's t test) in vaginal CFU counts between (a)untreated or SP-treated rats, and (b) KM-treated or fluconazole-treatedrats. There was no significant difference between untreated andSP-treated rats, and there was no significant difference betweenKM-treated and fluconazole-treated rats.

KM significantly accelerates the early rate of clearance (1-5 days) ofthe fungus from rat vagina, also providing a substantial resolution ofthe infection (less than 10³ CFU/ml of vaginal fluid) within three weeksfrom challenge, when the untreated controls still had from 2 to 4×10⁴Candida CFU/ml of vaginal fluid. No acceleration of the fungal clearanceand no effect on resolution of infection was provided by SPadministration. The therapeutic benefit of KM was substantiallycomparable with that of fluconazole.

Results for both test strains over 28 days were as follows: C. albicansvaginal CFU (×10³) (±SD) on days: Experimental group 0 7 14 28 1. C.albicans SA-40, >100  98 ± 12 42 ± 7 38 ± 6 no treatment 2. C. albicansSA-40 + >100 13 ± 4  2 ± 1 <1 50 μg fluconazole 3. C. albicansSA-40 + >100 12 ± 7  1 ± 1 <1 KM 4. C. albicans AIDS 68, >100  70 ± 1055 ± 8 18 ± 3 no treatment 5. C. albicans AIDS >100 64 ± 6 48 ± 3 12 ± 468 + 100 μg fluconazole 6. C. albicans AIDS >100 40 ± 5 18 ± 2 <1 68 +KM

On day 7, 14 and 28, differences in CFU vaginal counts werestatistically significant (p<0.05, two-tailed Student's t test) between:

-   -   group 1 and group 2    -   group 1 and group 3    -   group 4 and group 6    -   group 1 and group 5    -   group 1 and group 6    -   group 5 and group 6

There was no statistically significant difference between:

-   -   group 2 and group 3    -   group 4 and group 5

In contrast to treatment with 100 μg fluconazole, KM had a therapeuticeffect also in rat vaginal infection caused by the fluconazole-resistantC.albicans strain.

In Vivo Anti-Candida Activity of KM in Systemic Infection Model

KM was tested using a well-established rapidly-lethal systemic mousemodel [72] of C. albicans infection. Groups of 8 Balb/C female mice(weight 18-21 g) were challenged with 5 LD₅₀ (10⁷ cells of SA-40) by theintravenous route. 50 μg KM were administered intraperitoneally forthree days starting on day 0 (i.e. 1 hour after the fungal challenge)and at 24 and 48 hours thereafter. Controls were untreated animals oranimals treated with peptide SP (same dosage and treatment schedule asthose treated with KM). The animals were then followed for mortality andinternal organ invasion for 60 days. Any beneficial effect wasestablished in term of prolongation of the median survival time (indays) and reduction of total mortality. Assessment by necroscopy showedthat the death of the animals was due to the fungus, and assessment ofinternal organs showed invasion by C.albicans.

In parallel experiments, SCID mice were used instead of immunocompetentmice in order to verify whether the curative effect of KM required theparticipation of host adaptive immunity. These experiments used the samechallenging fungal burden, schedule of KM and SP treatments, and C.albicans-caused mortality end-points as described above.

In all experiments, KM exerted a similar beneficial therapeutic effectin terms of mortality delay and animal cure (60 days). As a typicalexample, FIG. 9 shows the Kaplan-Meyer survival curve of SCID micechallenged with 5×LD50 of C.albicans and treated with 50 μg of eitherKM, fluconazole or SP, or left untreated. KM was seen to increase themedian survival time from 1 day of the untreated control to >60 days. Inaddition, only ⅛ KM-treated animals died as compared to 8/8 deaths inuntreated mice or those treated with SP. In all cases, death wasattributable to C.albicans challenge as shown by fungus burden in thekidneys. FIG. 9 shows that KM out-performed fluconazole.

Blocking of KM Activity by Glucans

As shown above, KM shows anti-candida activity. As KM isdistantly-related to the KT antibody, which interacts with cell surfaceβ-glucans, the possible involvement of glucans in KM's activity wasinvestigated.

The binding of the KT mAb to germinating cells of C.albicans wasassessed by immunofluorescence [12]. KM completely inhibited the bindingof KT to the cells, whereas SP did not.

In further experiments, the CFU assay was performed as before, using25μg/ml KM or SP, except that various concentrations (between 12.5μg/mland 100 μg/ml) of either laminarin (β-1,3-glucan, from Sigma) orpustulan (β-1,6-glucan, from Calbiochem) were included. Results were asfollows: Conc^(n) Glucan (μg/ml) CFU with KM CFU with SP None — 02822.00 ± 122.00 Laminarin 12.5 0 2525.33 ± 92.72 Laminarin 25  1.33 ±0.58 2780.00 ± 264.21 Laminarin 50  115.33 ± 12.58 2742.66 ± 105.09Laminarin 100 2766.66 ± 63.54 2740.00 ± 282.13 Pustulan 12.5 0 2380.00 ±148.04 Pustulan 25 0 2500.00 ± 52.00 Pustulan 50 0 2666.00 ± 306.49Pustulan 100 0 2824.00 ± 119.82

Thus the candidacidal activity of KM was strongly and dose-dependentlyinhibited by laminarin, but not by pustulan.

These data suggest that the candidacidal KM decapeptide competes for thebinding site of KT-IdAb on fungal cell wall, and the receptor seems tocontain β-1-3 glucans.

Anti-A.Castellani Activity

In addition to their anti-candida activity, the KM peptide and the K20monoclonal antibody were tested for activity against trophozoites (theinfectious form) of the eukaryotic free-living soil amoeba Acanthamoebacastellanii —a cause of encephalomyelitis and keratitis which can causesevere ocular inflammation and visual loss.

A.castellani was grown in PYG medium at 37° C. or 25° C., in thepresence of either SP or KM peptides at 1 μg/ml, 10 μg/ml or 100 μg/ml(or, as a control, with no peptide). The effect on growth over six days,in terms of the number of trophozoites, is shown in FIG. 14. As seen inFIGS. 14C & 14D, the scramble peptide SP has no antimicrobial activityat either 37° C. or 25° C., whereas KM (FIGS. 14A & 14b) shows goodantimicrobial activity.

FIG. 15 shows the in vitro activity of KM at 190 μg/ml on cell viabilityof A.castellanii evaluated at 25° C. in comparison with SP. FIG. 15Ashows a reduction in cell viability (p<0.05) after six hours ofco-incubation. FIG. 15B shows a similar reduction (p<0.05) after sixhours co-incubation followed by 18 hours incubation in fresh PYG medium.

The inhibitory effect of monoclonal antiidiotypic antibody K20, testedover 6 days at 370° C. in the same way as described for KM, is shown inFIG. 16. The effect can be compared to that of the rat monoclonal K10(FIG. 17). After 6 days of incubation, the number of trophozoites perwell using K20 was lower than when using K10 at 25 μg (2700 vs. 3100)and 50 μg (2800 vs. 3080).

Range of KM Activity

KM shows potent anti-C.albicans and anti-A.castellani activity. KM wasalso found to be effective against other microorganisms which are veryimportant from an epidemiological point of view, such as multidrugresistant strains of Candida spp. and Mycobacterium tuberculosis,Cryptococcus neoformans (FIG. 10) and Aspergillus fumigatus, but alsoagainst methicillin-resistant strains of Staphylococcus aureus (FIG.11), and penicillin-resistant strains of Streptococcus pneumoniae.

Surprisingly, KM was also found to have anti-viral activity againstinfluenza A virus and HIV-1.

Influenza A Virus

The effect of KM on influenza virus replication was compared to thescramble peptide SP control. As a further control, replication inmaintenance medium alone was tested.

Two different strains of type A influenza virus (Ulster/73/H7N1, avian;NWS/33/H1N1, human neurovirulent) have been previously demonstrated toefficiently replicate in LLC-MK2 (Rhesus monkey kidney), MDCK (MadinDarby Canine Kidney) and AGMK-37RC (African Green Monkey Kidney) cellcultures. Confluent monolayers of the different cell lines were infectedwith the virus (moi=20 pfu, higher than a normal in vivo infection) inpre-warmed PBS (pH 7.4). After a 40 minute adsorption period,maintenance medium (MM) containing either KM or SP was added to the cellcultures and virus titre was determined after 24, 36, 48 or 72 hours ofinfection by haemagglutinin (HA) titration in triplicate samples on cellsupernatant, after centrifugation at 2,000×g to remove cellular debris.

The effect of KM and SP on HA titres 24 hours after infection of LLC-MK2cells by Ulster/73 is shown in FIG. 18. At 50μg/ml or below, KM and SPhad little or no effect on viral growth. Above 60μg/ml, however, KMpeptide interferes with viral growth, with complete blocking at 80μg/ml. Similar effects were seen for the other virus, with the othercell lines, and at the other time points. Thus KM can suppress viralreplication in a dose-dependent manner.

In a different set of experiments, fresh MM containing either KM or SPwas added to the cells at time 0 and then substituted with the samepeptide/medium mixture every 12 hours starting at 24 hours and ending at72 hours. KM at 80 μg/ml was able to completely block viral productionat all time points tested. Using SP, viral titres were similar to thoseobtained by growth in MM only.

The effect of KM on influenza virus replication was also tested usinghaemadsorption assays. HA is synthesised during replication and insertedinto the cell membrane before viral budding. Haemadsorption of red bloodcells to infected cells correlates with the integrity of theglycosylated HA as well as with its correct insertion into the cellmembrane.

Cell cultures were infected, treated with 80 μg/ml of either KM or SP,as described above, and haemadsorption assays were carried out after 48hours. A significant reduction of OD_(420nm) was observed in infectedcells treated with KM, demonstrating a significant reduction of viral HAmolecules on the plasma membrane of those cells. Neither KM nor SPinterferes with virus-specific receptors on red blood cells (RBC).

Finally, the effect of laminarin (β-1,3-glucan) on anti-influenzaactivity was determined. As shown above, Laminarin interferes with KM'scandidacidal activity. KM or SP (80 μg/ml) was mixed with laminarin (320μg/ml) and the mixture was added to infected cells at time 0, withoutpreincubation or after 10 minutes of preincubation. Similar HA titreswere obtained after 24 hours using KM/laminarin and SP/laminarin,suggesting that laminarin abolishes the antiviral activity of KM. Thiseffect was not seen with pustulan.

HIV-1

The effect of KM on HIV-1 replication was compared to the scramblepeptide SP control. HIV-1 can replicate in peripheral blood mononuclearcell (PBMC) cultures in the presence of exogenous IL-2 [73]. To test KMactivity, PBMC cultures were obtained from patients in acute infectionphase in whom HIV-1 proviral load was known.

PBMCs were cultured in 48-well plates at the concentration of 10⁶cells/ml in RPMI 1640 medium supplemented with 10% FCS in the presenceof 50 U/ml of rIL-2. Half of the culture medium was added at day 8.Supematants and cells were harvested for analysis of HIV RNA andproviral contents, respectively. Exogenous rIL-2 was added every 3 to 4days, KM- or SP-peptide was added every 7 days. HIV-1 expression wasdetermined using HIV RNA branched kit (b-DNA, Bayer™), whereas HIV-1proviral load was determined using gene-detective HIV-1 gag EOA kit(ZeptoMetrix™). For phenotype determination, cells were analysed by flowcytometry after staining with mAbs directed to CD antigens.

After PBMC isolation, the majority of PBMC of HIV cultures wereT-lymphocytes, as shown by the expression of CD3 Ag (CD4+=41%;CD8+=36%). All primary PBMC cultures remained viable apparently for atleast 20 days. In the presence of exogenous IL-2, all CD3+T-cellsexpressed CD25 and HLA-DR cell surface activation markers after 7 daysof culture.

Primary cultures were established from the patient's cryo-preserved PBMCin the presence of 1× and 10× concentration of KM peptide (1 μg and 10μg, respectively). As controls, SP peptide was used at the sameconcentrations. Other control cultures (untreated cells) received mediumonly.

The kinetics of HIV RNA production at different time points of PBMCcultures are shown in FIG. 19. The cultures showed early peaks of viralcopies within 5 to 10 days of culture which then decreased incorrespondence to the HIV-1-induced loss of CD4+ cells. HIV replicationin these PBMC cultures was considerably lower in the presence of KM(<44%, mean), with both concentrations of KM having similar effects onthe levels and kinetics of HIV RNA production.

SP-treated cells showed the same levels and kinetics of HIV RNAproduction as untreated controls.

D-Amino Acid Derivatives of KM and KM0

KM0 and KM were synthesised using the same amino acids residues, but inthe D- rather than L-conformation (KM6 and KM7). Scramble peptidecontrols SP0 and SP were also synthesised using D-amino acids.

KM6 and KM7 each showed candidacidal activity in the CFU assay (FIG.12). Activity was slightly lower than the L-amino acid polypeptides, butthis in vitro reduction does not take into account the in vivo increasein half life which would be expected.

A D-amino acid polypeptide with both the Glu→Ala and Cys→Sersubstitutions (SEQ ID 33) is useful.

Toxicity

Toxicity of KM was assessed by in vitro incubation with LLC-MK2 rhesusmonkey kidney cells. The cells were maintained in Eagle's Minimumessential Medium (MEM) supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100 μg/ml streptomycin at 37° C. in a humidifiedatmosphere containing 5% CO₂. The cells were plated in triplicate in6-well dishes at 4×10⁴ cells per well and cultured for 24 hours. Serialdilutions of peptides (final concentrations between 0 and 500 μg/ml) inmedium containing 10% fetal bovine serum were then added to cells andincubated for 24 hours at 37° C. Cells were subsequently treated withMTT (50 μg per well) and incubated for another 2 hours. Aftersolubilisation of the formazan dye in DMSO, the absorbance of each wellwas measured at 550 and 620 nm. Peptide SP was also tested.

Cell viability as T/C % where T represents the mean absorbance of thetreated cells of the controls (FIG. 20).

The peptide displayed no toxic effects, even at 500 μg/ml.

Equivalent Peptides From Within H20

An alignment of H6 and H20 sequences is given below, with CDRs in bold:        10        20        30        40        50        60        70         |         |         |         |         |         |         |H6MAQVKLQESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGD-GSTDYNSALKSRL****:**:**. ** .. *:.::**.***.:..* ::**:* * :****:* *  : *.*:* . ::.:H2OMAQVQLQQSGAKLVRSGASVKLSCTTSGFNIKDYYMHWVKQRPEQGLEWIGWIDPENGDTEYAPKFQGKT        80        90       100       110       120       130       140         |         |         |         |         |         |         |H6SISKDNSKSQVFLKMNSLQTDDTARYYC-------------LYAMDYWGQGTTVTCSSGGGGSGGGGSGG::: *.*.. .:*::.** ::*** ***              ****************************H2OTLTADTSSNTAYLQLSSLTSEDTAVYYCNAWVYDGYSGDFYYYAMDYWGQGTTVTCSSGGGGSGGGGSGG       150       160       170       180       190       200       210         |         |         |         |         |         |         |H6GGSDIELTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSG************::*********:***********:*:**** :*** *** ******************H2OGGSDIELTQSPAIVSASPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSG       220       230       240       250       260         |         |         |         |         | H6TSYSLTISSMEAEDAATYYCQQWSSNPYTFGGGTKLEIKRAAAGAPVPYPDPLEPR ********************* ** * *** ************************ H2OTSYSLTISRMEAEDAATYYCQQRSSYPLTFGAGTKLEIKRAAAGAPVPYPDPLEPR

H20 peptide sequences corresponding to H6 peptides KM, KM0 , KM1, KM2,KM4 and KM5 are: H6 KM0 KM1 KM2 KM4 KM5 KM H6 SEQ ID 3 23 24 26 27 4 H20SEQ ID 34 35 36 37 38 39

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES (the contents of which are hereby incorporated in full)

[1] Magliani et al. Clin. Microbiol. Rev., 10:369-400, 1997

[2] Polonelli et al. J. Clin. Microbiol., 24:866-869, 1986

[3] Aliouat et al. Serodiagn. Immunother. Infect. Dis., 5:102-106, 1993

[4) Conti et al. J. Infect. Dis., 177:807-811, 1998

[5] Polonelli & Morace J. Clin. Microbiol., 25:460-462, 1987

[6] Polonelli & Morace J. Clin. Microbiol., 26, 602-604, 1988

[7] Polonelli et al. Scan. J. Immunol., 37:105-110, 1993

[8] Polonelli et al. J. Immunol., 152:3175-3182, 1994

[9] Séguy et al., FEMS Immunol. Med. Microbiol., 22:145-149, 1998

[10] Polonelli et al., J. Immunol. 156:1880-1885, 1996

[11] Séguy et al. Mol. Med., 3:544-552, 1997

[12] Polonelli et al., Clin. Diagn. Lab. Immunol., 4:142-146,1997

[13] Magliani et al., Nature Biotechnol., 15:155-158, 1997

[14] Cenci et al. Infect Immun 2002 May; 70(5):2375-2382

[15] Conti et al., Mol. Med., 6/7:613-619, 2000

[16] Séguy et al., J.Euk. Microbiol., 44:37S, 1997

[17] Breedveld (2000) Lancet 355(9205):735-740.

[18] Gorman & Clark (1990) Semin. Immunol. 2:457-466

[19] Jones et al. Nature 321:522-525 (1986)

[20] Morrison et al., Proc. Natl. Acad. Sci, US.A., 81:6851-6855 (1984)

[21] Morrison & Oi, Adv. Immunol., 44:65-92 (1988)

[22] Verhoeyer et al., Science 239:1534-1536 (1988)

[23] Padlan, Molec. Immun. 28:489-498 (1991)

[24] Padlan, Molec. Immunol. 31(3):169-217 (1994).

[25] Kettleborough et al., Protein Eng. 4(7):773-83 (1991).

[26] WO 98/24893

[27] WO 91/10741

[28] WO 96/30498

[29] WO 94/02602

[30] U.S. Pat. No. 5,939,598.

[31] Goletz et al. J Mol Biol 2002 Feb 1;315(5): 1087-97

[32] Morrison & Weiss (2001) Curr. Opin. Chem. Biol. 5:302-207.

[33] Weiss et al. (2000) PNAS USA 97:8950-8954.

[34] Carter (1994) Methods Mol Biol 36:207-223.

[35] Li & Roller (2002) Curr Top Med Chem 2:325-341.

[36] Bodanszky (1993) Principles of Peptide Synthesis (ISBN:0387564314).

[37] Fields et al. (1997) Methods in Enzymology 289:Solid-Phase PeptideSynthesis. ISBN: 0121821900

[38] Chan & White (2000) Fmoc Solid Phase Peptide Synthesis ISBN:0199637245.

[39] Kullmann (1987) Enzymatic Peptide Synthesis. ISBN: 0849368413.

[40) Ibba (1996) Biotechnol Genet Eng Rev 13:197-216.

[41] Strugnell et al. (1997) Immunol Cell Biol 75(4):364-369.

[42] Robinson & Torres (1997) Seminars in Immunol 9:271-283.

[43] Donnelly et al. (1997) Annu Rev Immunol 15:617-648.

[44] Brunham et al. (2000) J Infect Dis 181 Suppl 3:S538-43.

[45] Svanholm et al. (2000) Scand J Immunol 51(4):345-53.

[46] Beninati et al. (2000) Nature Biotechnology 18:1060-1064.

[47] Bevan & Makower (1963) pages 202-203 of Geerts (ed.), Geneticstoday, XIth International Congress on Genetics vol. 1. Pergamon Press,Oxford, England.

[48] Kazmierski (1999) Peptidomimetics Protocols. ISBN: 0896035174.

[49] Abell ( 1999) Advances in Amino Acid Mimetics and Peptidomimetics.ISBN: 0762306149.

[50] U.S. Pat. No. 5,331,573 (Balaji).

[51] Goodman et al. (2001) Biopolymers 60:229-245.

[52] Hruby & Balse (2000) Curr Med Chem 7:945-970.

[53] Ribka & Rich (1998) Curr Opin Chem Biol 2:441-452.

[54] Chakraborty et al. (2002) Curr Med Chem 9:421-435.

[55] Computer-Assisted Lead Finding and Optimization (eds. Testra &Folkers, 1997).

[56] Available from Molecular Simulations Inc (http://www.msi.com/).

[57] Davic & Lawrence (1992) Proteins 12:31-41.

[58] Caflish et al. (1993) J Med. Chem. 36:2142-67

[59] Eisen et al. (1994) Proteins: Str. Funct. Genet. 19:199-221.

[60] Böhm (1992) J Comp. Aided Molec. Design 6:61-78.

[61] Gehlhaar et al. (1995) J Med. Chem. 38:466-72.

[62] Moon & Howe (1991) Proteins: Str. Funct. Genet. 11:314-328.

[63] Available from http://chem.leeds.ac.uk/ICAMS/SPROUT.html.

[64] Lauri & Bartlett (1994) Comp. Aided Mol. Design 8:51-66.

[65] Available from Tripos Inc (http://www.tripos.com).

[66] Rotstein et al. (1993) J. Med Chem. 36:1700.

[67] Lai (1996) J Chem. Inf. Comput. Sci. 36:1187-1194.

[68] Gennaro (2000) Remington: The Science and Practice of Pharmacy.20th ed., ISBN: 0683306472

[69] Almeida & Alpar (1996) J Drug Targeting 3:455-467.

[70] Wills et al. (2000) Emerging Therapeutic Targets 4:1-32.

[71] De Bemardis et al., in Handbook of animal models of infection (eds.Zak, O. & Sande, M.A.) 735-740, Academic Press, New York, 1999

[72] Mencacci et al., Infect. Immun., 62:5353-5360, 1994

[73] Casoli et al. (2000) Blood 95: 2760-2769.

1. A polypeptide comprising: at least one amino acid sequence which is afragment of at least x amino acids from the amino acid sequence of avariable region of an anti-idiotypic antibody which recognizes theidiotope of an antibody specific for a yeast killer toxin, optionallywith y amino acids within said x amino acids being substituted bydifferent amino acid(s), wherein x is at least 3 and y is at least
 1. 2.The polypeptide of claim 1, consisting of at least 3 amino acids.
 3. Thepolypeptide of claim 1 consisting of at most 90 amino acids.
 4. Thepolypeptide of claims consisting of between 8 and 20 amino acids.
 5. Thepolypeptide of claim 4, consisting of 10 amino acids.
 6. The polypeptideof claim wherein x is at least
 8. 7. The polypeptide of claim 6, whereinx is
 10. 8. The polypeptide of claim wherein at least 1 amino acidwithin said x amino acids is substituted by different amino acid(s). 9.The polypeptide of claim 1, wherein the fragment of at least x aminoacids preferably includes at least 1 amino acid from a CDR within theantibody.
 10. The polypeptide of claim 1, comprising L amino acidsand/or D amino acids.
 11. The polypeptide of claim comprising sequenceAA₁-AA₂-AA₃-AA₄-AA₅-AA₆-AA₇-AA₈-AA₉-AA₁₀, wherein: each of AA₁ . . .AA₁₀ is independently a D- or L- amino acid; AA₁ is E, A or G; AA₂ is K,A or G; AA₃ is V, A or G; AA₄ is T, A or G; AA₅ is M, A or G; AA₆ is T,A or G; AA₇ is C, S, A or G; AA₈ is S, A or G; AA₉ is A or G; and AA₁₀is S, A or G; provided that no more than 4 of AA₁, AA_(2,) AA_(3,)AA_(4,) AA_(5,) AA_(6,) AA_(7,) AA_(8,) AA_(9,) and AA₁₀ are A; andprovided that no more than 2 of AA₁, AA_(2,) AA_(3,) AA_(4,) AA_(5,)AA_(6,) AA_(7,) AA_(8,) AA_(9,) and AA₁₀ are G.
 12. The polypeptide ofclaim 1, comprising amino acid sequence SEQ IDs: 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or71, with constituent amino acids in the D- and/or L- configuration. 13.The polypeptide of claim 12, consisting of SEQ ID 3, 4, 23, 27 or 33.14. A polypeptide consisting of SEQ ID 4 or SEQ ID 33, and consisting ofD-amino acids.
 15. A microbicidal peptidomimetic compound which isisosteric with respect to the polypeptide of claim
 1. 16. Ananti-idiotypic antibody which recognizes the idiotope of an antibodyspecific for a yeast killer toxin, with the proviso that the antiidiotypic antibody is not the K10 rat monoclonal antibody.
 17. Anantibody which recognizes the idiotope of an antibody of claim
 16. 18.The antibody of claim 16 wherein the antibody is a single-chain Fvantibody.
 19. The antibody of claim 18, wherein the antibody is H6 orH20.
 20. The antibody of claim 16, wherein the antibody is a monoclonalantibody.
 21. The antibody of claim 20, wherein the antibody is a humanor humanized antibody.
 22. The antibody of claim 21, wherein theantibody comprises one or more CDR from H6, H20 or K20.
 23. The antibodyof claim 16 wherein the toxin is the killer toxin from P. anomala.
 24. Apolypeptide comprising: at least one amino acid sequence which is afragment of at least x amino acids from the amino acid sequence of avariable region of an antibody of claim 16, optionally with y aminoacids within said x amino acids being substituted by different aminoacid(s), wherein x is at least 3 and y is at least
 1. 25. Nucleic acidthat encodes a polypeptide of claim 1, and/or an antibody whichrecognizes the idiotope of an antibody specific for a yeast killertoxin, with the proviso that the anti idiotypic antibody is not the K10rat monoclonal antibody.
 26. The antibody of claim 16 or the polypeptidecomprising: at least one amino acid sequence which is a fragment of atleast x amino acids from the amino acid sequence of a variable region ofan anti-idiotypic antibody which—recognizes the idiotope of an antibodyspecific for a yeast killer toxin, optionally with y amino acids withinsaid x amino acids being substituted by different amino acid(s), whereinx is at least 3 and y is at least 1 which has antimycotic activityand/or antibiotic activity. 27-34. (canceled)
 35. A pharmaceuticalcomposition comprising (a) the antibody of claim 16 and (b) apharmaceutical carrier.
 36. A pharmaceutical composition comprising (a)the polypeptide of claim 1 and (b) a pharmaceutical carrier.
 37. Apharmaceutical composition comprising (a) the peptidomimetic of claim 15and (b) a pharmaceutical carrier.
 38. A pharmaceutical compositioncomprising (a) the nucleic acid of claim 25 and (b) a pharmaceuticalcarrier.
 39. A method for treating a patient suffering from a microbialand/or viral infection, comprising administering to the patient thepharmaceutical composition of claim
 35. 40. A method for treating apatient suffering from a microbial and/or viral infection, comprisingadministering to the patient the pharmaceutical composition of claim 36.41. A method for treating a patient suffering from a microbial and/orviral infection, comprising administering to the patient thepharmaceutical composition of claim
 37. 42. A method for treating apatient suffering from a microbial and/or viral infection, comprisingadministering to the patient the pharmaceutical composition of claim 38.